Mar. 17, 1999 DURHAM, N.C. -- Duke University Medical Center researchers believe they have answered one of cancer's central enigmas: why some blood vessels are able to grow to, and feed, tumors, while other vessels are not. In the March 16 issue of the Proceedings of the National Academy of Science, the scientists report the blood protein angiostatin, which is known to stop the growth of new blood vessels to tumors, works by depleting the chemical energy that blood vessel cells need to grow.
To do this, angiostatin latches on to and inhibits ATPsynthase, an enzyme that provides chemical energy for the cell. Without that energy, blood vessels cannot grow to the site of a tumor, and without the nutrient supply in blood, tumors cannot grow larger than a pinhead.
Conversely, when unchecked by angiostatin, ATPsynthase provides a generator of sorts to blood vessels so that they can survive in the atmosphere of cell death caused by cancer. Cancer researchers have long wondered how these vessels stay vigorous enough to continue to grow to and feed tumors.
Lead scientists Dr. Sal Pizzo and Tammy Moser said the discovery is "startling" because the ATPsynthase enzyme has never before been found on the surface of endothelial cells, which are the cells that line blood vessels.
In fact, ATPsynthase has not been known to exist outside of a cell body. It has only been found within a mitochondria, a sac-like structure which acts as a cell's chemical powerhouse.
"It was surprising to see ATPsynthase on blood vessel cells because it was never expected there," said Pizzo in an interview. "But it makes perfect biological sense, and it's terribly exciting."
The finding offers both theoretical as well as practical implications, Pizzo said. It offers a novel insight into the body's use of ATPsynthase as "power packs" in situations where energy is depleted. And it suggests a new route to developing drugs that block angiostatin.
"Instead of using the whole angiostatin protein as a drug to stop tumor angiogenesis, as efforts are now underway to do, it may be possible to design a small molecule that will do what angiostatin does -- turn off ATPsynthase," Moser said.
Pizzo added that, in the future, it may be possible to find a molecule that turns on ATPsynthase "in cases where you want new blood vessel growth, such as in heart disease."
Because she led the laboratory investigation, Moser checked, rechecked and expanded her findings over a span of several years to be sure of the conclusions. "A lot of labs might have pulled out and said it was a mistake, but it seemed a very rational explanation worthy of replication," she said.
The discovery is very important because "the ATPsynthase-binding protein may be used as a target to find small molecules, which could mimic angiostatin, but could be taken orally, and perhaps would be easier to manufacture," according to Dr. Judah Folkman, a cancer researcher at Children's Hospital in Boston who pioneered angiostatin research. "If such small molecules are developed, they may also enhance the activity of angiostatin."
Folkman, a member of the National Academy of Sciences, was the scientist who submitted the paper to PNAS, the academy's journal, although he didn't participate in the research.
Working with Pizzo and Moser on the study were Duke researchers Iain Asplin, Jan Enghild and Lorraine Everitt. Their collaborators include Danish researcher Peter Hojrup, and, from Northwestern University Medical School, Sharon Stack, Susan Hubchak and William Schnaper. The study was supported by a research grant from Glaxo Wellcome Inc. Duke University holds the patent rights to the discovery.
The field of angiostatin research grew from the observation that some, but not all, tumors seem to be able to control the spread, or metastasis, of cancer elsewhere in the body.
Cancer physicians have long known that sometimes, when a single large tumor is removed from a patient, subsequent tumors will come back quickly and spread, seeding the body with deadly fast-growing tumors.
A decade ago, Folkman proposed that tumors themselves regulate the growth of blood vessels. One notion was that tumors could produce proteins that would travel through the bloodstream, preventing vessels from growing to any new cancer metastasis. This idea explains what has been observed, although no one understands why a tumor would exert such control, or why only some tumors work in this way.
Folkman's lab later found the substance that inhibits blood vessel growth. It was a small piece of a large and common blood protein called plasminogen, which is involved in blood clotting. He called this protein "angiostatin," and demonstrated it was involved in "anti-angiogenesis" -- stopping the process of new blood vessel growth.
Folkman then showed in animal experiments that injections of the angiostatin protein stopped tumors from growing, and efforts have been underway in the last year to produce a drug for human cancer therapy based on the angiostatin molecule.
Pizzo and Moser set out to study what happened when angiostatin "bound" on the surface of endothelial cells. They looked for where angiostatin's "key" fit into the cellular "lock" that then shut down vessel growth.
Since plasminogen is known to bind to a protein called annexin II on endothelial cells, they expected to find that angiostatin bound to same site. After several years of gathering material and analyzing data, Moser found a different molecule, which she identified by mass spectrometry as ATPsynthase. Moser then tested her findings by introducing an antibody to ATPsynthase that would block the action between angiostatin and endothelial cells. Indeed, she found the antibody blocked angiostatin's ability to inhibit proliferation by 90 percent.
The researchers stressed that while there is still much to learn about how angiostatin regulates blood vessel growth, there are now many new interesting theories to explore.
For example, Pizzo and Moser speculate that blood vessels grow into tumors by capitalizing on the death of cancer cells in the core of tumors due to lack of oxygen.
A tumor is an ever expanding knot of cancer cells, and its central core is often composed of cells that are dying from a lack of oxygen. When cells die, they release a depleted form of chemical fuel called ADP. And it is ADP that the ATPsynthase molecule uses to produce ATP, the body's high energy fuel. So it may be that "vessels are feeding on cell death to grow," Moser said. "This is a very novel strategy."
Folkman added: "Because ATP is necessary for cells to resist conditions of low oxygen, and because the enzyme to produce ATP is situated on the outer surface of endothelial cells, angiostatin, by binding to this enzyme could preferentially inhibit endothelial cells that are in the vascular bed of a tumor, where oxygen would be lower than elsewhere in the body."
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