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Laboratory study explains clinical promise
ANN ARBOR, Mich.- For nearly five years, doctors at the University of Michigan Comprehensive Cancer Center have noted promising cancer-slowing results from early clinical trials of a drug that lowers the level of copper in cancer patients' blood.
Now, new U-M laboratory research results are telling them exactly how that experimental drug works, and showing them its cancer-fighting potential on a cellular level. The findings, published in the current issue of the journal Cancer Research, have implications for the approach to cancer treatment known as anti-angiogenesis.
The paper describes how the drug - tetrathiomolybdate, or TM - keeps tumor cells from sending signals that spur the formation of new blood vessels. By keeping copper low and blocking the NFkB signaling pathway, the researchers believe, TM blocks the angiogenesis, or blood-vessel creation, that lets cancer grow and spread.
Angiogenesis is thought to be a common denominator for many kinds of cancer, allowing tumors to grow locally and to metastasize to the rest of the body. The U-M team explored TM's anti-angiogenic potential using four methods in mice and cells.
Specifically, they showed that TM suppressed the growth of tumors in mice implanted with cells from an aggressive form of human breast cancer; kept new blood vessels from forming in cancer-prone cultures of rat artery cells; squelched the release of a key signaling molecule known to spur blood vessel formation; and prevented the formation of tumors in mice specially bred to develop breast cancer.
"Taken together, these results support the initial findings of the clinical trials that have been done with TM, and indicate that copper reduction can inhibit tumor angiogenesis with minimal adverse effects," says senior author Sofia D. Merajver, M.D., Ph.D., associate professor of internal medicine and director of the U-M Breast and Ovarian Cancer Risk Evaluation Program. She notes that the copper reduction achieved with TM is far greater that what can be achieved through diet alone.
Merajver has helped lead clinical and laboratory investigations of TM for cancer at the U-M for several years, including a current Phase II trial for advanced breast cancer patients. TM is also being tried at the U-M and other centers in patients with prostate cancer, breast cancer, head and neck cancer, multiple myeloma, liver cancer, mesothelioma and other malignancies.
The drug was originally developed for medical use by George Brewer, M.D., the Morton and Henrietta Sellner Professor of Human Genetics at the U-M, to treat the excess copper levels caused by a rare genetic disorder known as Wilson's disease.
Made up of sulfur and molybdenum, TM latches on to copper in the blood, and to a protein called albumin, in a process called chelation. The three-part complex formed by this bonding is then eliminated by the body.
TM has saved the lives of dozens of Wilson's disease patients treated at the U-M Health System's General Clinical Research Center, ridding their bodies of copper that would have damaged their brains and livers, and eventually would have killed them.
Even as the Wilson's disease treatment at the U-M began to achieve success in the 1990s, research at the U-M and elsewhere started to uncover the role of copper in angiogenesis - both the normal process that goes on constantly in the body, and the uncontrolled angiogenesis seen in cancer. Researchers found that copper was important to various "growth factors" that are necessary to the organizing process by which cells become part of new blood vessels.
Spurred by these discoveries, Merajver and Brewer teamed up for laboratory studies of TM against cancer. This led to a Phase I trial in a group of late-stage patients with various forms of cancer, whose results were published in January, 2000 in the journal Clinical Cancer Research.
That trial was intended just to test TM's safety and ability to lower copper levels in cancer patients. But it showed evidence of tumor stabilization in a handful of patients whose copper levels were reduced to one-fifth their original levels for three months or more.
Since that time, U-M researchers have studied TM on parallel clinical and basic research tracks. Currently, dozens of patients are enrolled in various Phase II trials at the U-M Comprehensive Cancer Center.
Meanwhile, Merajver and her team have continued to pursue the basic laboratory research needed to show how TM produces its anti-angiogenesis effect.
In the new study, the team used two animal models of breast cancer - one in which mice were given cancer by the transfer of human inflammatory breast cancer cells, and one using mice that had been specially bred to ensure that nearly all would develop cancer in their first year.
The results were striking. The mice with the aggressive human breast cancer "xenografts" that received TM had their tumor size suppressed by 69 percent, compared with mice that did not receive TM. Mice that received TM showed only sparse blood vessels in their tumors.
As for the mice genetically "preprogrammed" for breast cancer, none of the ones given TM preventively developed tumors. This statistically significant disease-free survival ended, though, when the mice were taken off TM - all of them developed tumors within two weeks. Microscope studies showed that those given TM had "microtumors" in their breast areas, suggesting that the cancer had started to form but could not grow bigger without a blood supply.
The research also used two in vitro, or cell-culture, approaches. In one, rings cut from the aortas of rats (a kind of tissue that's likely to form new blood vessels) were bathed in a culture of inflammatory breast cancer cells.
In the other, the researchers inserted breast cell and breast cancer cell nuclei with a genetic sequence into that could only be transcribed, or "read", by the NFkB transcription factor - along with a snippet of DNA that produced a telltale glowing molecule whenever NFkB did its job. In the cancer cell culture, NFkB activity was 2.5 times greater than that in the breast cell culture, but when TM was added to the disk, NFkB activity went down almost twice as much in cancer cells as in normal cells.
When the researchers looked at the genes involved in making the proteins that actually make up the NFkB molecule, they found that TM treatment cut the production of those proteins significantly. And when they looked at the interleukin and growth factor molecules whose transcription NFkB usually controls, the levels were lower in the presence of TM too.
"It appears that TM exerts its anti-angiogenic action at least in part by restricting the release of factors that promote angiogenesis, and by suppressing NFkB activity," says Merajver. "This is potentially exciting from a clinical perspective because NFkB is involved in cancer's resistance to chemotherapy and radiation therapy. And, the suppressive effect we've seen suggests a promising role for TM as a chemopreventive agent, in people who carry alterations in genes that make them susceptible to cancer."
The research was funded in part by the National Institutes of Health, the Food and Drug Administration, the U-M Comprehensive Cancer Center and the American Cancer Society. The drug has been licensed to Attenuon, LLC, a San Diego biotechnology company.
Besides Merajver and Brewer, the study's authors are: lead author Quintin Pan, M.D., Celina Kleer, M.D., Kenneth van Golen, Ph.D., Diane Robins, Ph.D., Jennifer Irani and Kristen Bottema, all of the University of Michigan Comprehensive Cancer Center; Robert D. Dick of the U-M Medical School Department of Human Genetics; and Carlos Bias, Magda de Carvalho, and Enrique Mesri of the Weill Medical College of Cornell University.