Four Genes Integral to Breast Cancer Metastasis in Mice

NEW YORK, April 11 -- Four genes drive the spread of breast cancer to the lungs and -- at least in mice -- their activity can be blocked by available drugs, found researchers here.

The four genes collaborate to build blood vessels at the primary tumor site, release tumor cells into the circulation, and help them breach lung capillaries to establish new tumors, said Joan Massagu, Ph.D., of Memorial Sloan-Kettering Cancer Center.

In a series of experiments in mice, Dr. Massagu and colleagues found that blocking the activity of any of the genes slowed tumor progression and metastasis, but blocking all of them brought the process almost to a halt.

The finding opens the way to new cancer treatments that prevent metastasis, rather than attempting to stop tumor cell replication, they said in the April 11 issue of Nature.

"We found that depriving aggressive metastatic tumor cells of these genes decreased both their ability to grow large aggressive tumors in the mouse mammary gland and also the ability to release cells from these tumors into the circulation," Dr. Massagu said.

"The remarkable thing was that while silencing these genes individually was effective, silencing the quartet nearly completely eliminated tumor growth and spread," he added.

The four genes code for the epidermal growth factor receptor ligand epiregulin, the cyclooxygenase Cox-2, and the matrix metalloproteinases 1 and 2, the researchers said.

Using a technique called RNA interference, they created human breast cancer cells that expressed only a few or none of the four genes. Then the various cell lines were injected into the mammary fat pads of mice.

Compared with mice who got unaltered cancer cells, all of the "knockdown" lines produced significantly smaller primary tumors (at P<0.05 and P<0.01, depending on what genes or gene combinations were blocked.)

But the cell line with all four genes silenced produced almost no tumor growth at all - less than 50 cubic millimeters after more than 40 days, compared with more than 650 for controls and about 150 for the next most effective knockout combination. Compared with controls, the difference was significant at P<0.001.

Analysis of the tumors showed they were deficient in new blood vessels, and what blood vessels they had were significantly shorter and less elaborate than those in the wild-type tumor.

To see what happened in the lungs, the researchers injected the various cell lines intravenously. In this experiment, blocking the genes individually had little effect on the growth of lung tumors.

However, silencing them in combination slowed tumor growth in the lungs, with the four-gene knockdown having the strongest effect, the researchers said.

A key factor was that tumor cells with the four-gene knockdown seemed to be trapped inside the lung capillaries, unable to escape through the wall of the blood vessels to begin forming tumors.

"When these cells reached the lung capillaries, they just got stuck there," Dr. Massagu said. "We concluded that metastatic cells use these same genes to loosen up cells in capillaries, so that the cells can penetrate the lung tissue to grow there."

Finally, Dr. Massagu and colleagues said, they tested the effect of blocking the four genes using drugs - cetuximab (Erbitux), which blocks the action of epiregulin, the Cox-2 inhibitor celecoxib (Celebrex) and the investigational matrix metalloproteinase inhibitor GM600125.

Individually, the drugs did little to slow the growth of mammary tumors, but the combination of all three had much the same effect as blocking all four genes - tumor growth was significantly slowed (P<0.001) compared with mice given a placebo injection.

As with the gene knockdown study, the drug treatment also trapped cancer cells in the lung capillaries and prevented the formation of new tumors, the researchers said. When the drugs were stopped, the cells regained the ability to penetrate the blood vessel walls, and new tumors were seen.

The experiments represent a "technological milestone" in the ability to create stable cell lines with several genes silenced, said Gerhard Christofori, Ph.D., of the University of Basel, in an accompanying News & Views article.

But many questions remain, Dr. Christofori said, including what other cells and factors are involved, whether the role of the four genes is specific to lung metastasis of breast cancer or applies to other sites, and whether the same genes drive the spread of other types of cancer.

And a key question, he said, is whether the "combinatorial treatment, which has proved so successful in the preclinical setting reported by these authors, (can) be further developed for clinical application."