Genetic 'Gang of Four' drives spread of breast cancer

Studies of human tumour cells implanted in mice have shown that the abnormal activation of four genes drives the spread of breast cancer to the lungs.

The new studies by Howard Hughes Medical Institute researchers reveal that the aberrant genes work together to promote the growth of primary breast tumours. Co-operation among the four genes also enables cancerous cells to escape into the bloodstream and penetrate through blood vessels into lung tissues.

Although shutting off these genes individually can slow cancer growth and metastasis, the researchers found that turning off all four together had a far more dramatic effect on halting cancer growth and metastasis.

Metastasis occurs when cells from a primary tumour break off and invade another organ. It is the deadliest transformation that a cancer can undergo, and therefore researchers have been looking for specific genes that propel metastasis.

In the newly published experiments, the researchers also found that they could reduce the growth and spread of human breast tumours in mice by simultaneously targeting two of the proteins produced by these genes, using drugs already on the market. The researchers are exploring clinical testing of combination therapy with the drugs-cetuximab (trade name Erbitux) and celecoxib (Celebrex)-to treat breast cancer metastasis.

The research team, led by Howard Hughes Medical Institute investigator Joan Massague at the Memorial Sloan-Kettering Cancer Center, published its findings in articles in the April 12 issue of Nature and in the online early edition of the Proceedings of the National Academy of Sciences on April 9.

In an earlier study, Massague and his colleagues had identified 18 genes whose abnormal activity is associated with breast cancer's ability to spread to the lungs. In the new study published in Nature, Massague and his colleagues at Sloan-Kettering, along with researchers from Hospital Clinic de Barcelona and the Institute for Research in Biomedecine in Spain, focused on four of these genes.

These genes, which code for epiregulin, COX2 and matrix metalloproteinases 1 and 2, were already known to help regulate growth and remodelling of blood vessels, Massague said.

"Our understanding of the genes for these four proteins and their behaviour in metastasis led us to hypothesise that they might be co-operating with each other in a way that would give an advantage to cells in the primary tumour," Massague said. "These same genes, we believed, might also be used for some related purpose in the target organ, the lung."

To test this idea, the researchers silenced various combinations of the four genes in human breast cancer cells that had metastasised to the lung using RNAi, and then tested these cells in mice.

"We found that depriving aggressive metastatic tumour cells of these genes decreased both their ability to grow large aggressive tumours in the mouse mammary gland and also the ability to release cells from these tumours into the circulation," he said. "The remarkable thing was that while silencing these genes individually was effective, silencing the quartet nearly completely eliminated tumour growth and spread."

Microscopic analysis of blood vessel structure in the tumours revealed that knocking down all four genes greatly reduced growth of the tangle of blood vessels typically seen in tumours. Further experiments revealed that the tumour blood vessels that did form allowed fewer cancer cells to escape into circulation.

The researchers next explored how loss of the four abnormal genes affected the metastatic capability of the cells in the lung. They injected cells deficient in the four genes directly into the circulatory system of mice.

"When these cells reached the lung capillaries, they just got stuck there," Massague 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.

"These findings provide a beautiful explanation for how the genes that we identified in breast cancer patients as being associated with lung metastasis manipulate blood vessels to give them an advantage both in the primary tumours and in the lung."

Two drugs already on the market act directly on proteins produced by the genes Massague's group had been studying. Cetuximab is an antibody that blocks the action of epiregulin and is used to treat advanced colorectal cancer. Celecoxib is an inhibitor of COX2 that is used as an anti-inflammatory, and is being tested in clinical trials against many types of cancer. The researchers also tested whether cetuximab and celecoxib would work effectively in concert to reduce metastasis in mice.

"We found that the combination of these two inhibitory drugs was effective, even though the drugs individually were not very effective. This really nailed the case that if we can inactivate these genes in concert, it will affect metastasis."

He said that while clinical trials of the drug combination are being discussed, "there are already treatments to diminish the chance of metastasis in breast cancer, so such trials would have to be designed very carefully to understand how and whether the new drug combination would be of additional benefit."

In the article published in the Proceedings of the National Academy of Sciences, Massague and his colleagues explored how the entire group of 18 genes, called the 'lung metastasis gene-expression signature' (LMS) influenced both breast tumour growth and spread to the lungs. Co-authors on the paper were from the University of Chicago, The Netherlands Cancer Institute, Veridex L.L.C., The Cleveland Clinic and the Erasmus Medical Center in The Netherlands.

"There has been an undeniable link between tumour size and growth and metastatic risk, but the molecules and mechanisms underlying this link have remained unresolved," Massague said. "The hypothesis we wanted to test was that these signature genes play a role in both primary tumour growth and metastasis to the lung."

After analysing 738 human breast cancer tumours, the researchers concluded that those in which the LMS genes were abnormally active were, indeed, more likely to develop lung metastases. They also found that the activity of these LMS genes gave cancer cells a growth advantage by allowing tumours to develop a rich network of blood vessels to deliver oxygen and nutrients.

Although large tumours are more likely to metastasise, Massague said his group's findings indicated that the activity of the LMS genes was also critical to the metastasis process. "As the tumours grow and become enriched with LMS-positive cells, because the genes give them an advantage, they reach a point where the tumour becomes richly vascularised. Then, they can massively execute the advantage the LMS genes provide them to metastasize to the lung."

Massague said he and his colleagues will explore in more detail the function of other LMS genes, in addition to the four reported in the Nature paper. They plan to investigate whether shutting down other LMS genes will affect metastasis of breast cancer to the lung, and whether the LMS genes influence breast cancer metastasis to other sites, such as the bone and brain. Finally, they will explore whether the LMS genes play a corresponding role in metastasis of other cancers -- such as sarcoma, melanoma and colon cancer -- to the lung.

Source: Howard Hughes Medical Institute