ddn Cancer Research News Exclusive: Mapping metastasis
BALTIMORE—Even in the face of an assortment of increasingly effective treatments, breast cancer continues to claim several thousand lives each year, ranking as the third most deadly type of cancer. The primary cause for breast cancer's high death rate is metastasis, but despite its importance, few specifics are known regarding the process.
Thanks to a recent study from a team of researchers at Johns Hopkins, however, there are a few more answers. The team's work revealed how a specific protein that ensures cell survival in low-oxygen situations can cause cancer cells to spread into the lymphatic system, with the results published in the Sept. 10 edition of the Proceedings of the National Academy of Sciences Online Early Edition.
The study focused primarily on a protein known as hypoxia-inducible factor 1, or HIF-1. In the case of solid tumors, in which cancer cells can grow so densely that they starve themselves of oxygen, cancer cells will activate this protein to initiate the growth of new blood vessels. The HIF-1 protein was discovered nearly 20 years ago by Gregg Semenza, M.D., Ph.D., director of the vascular program at Hopkins' Institute for Cell Engineering, and his team, as noted in a Johns Hopkins press release.
Semenza says that HIF-1 has been studied by numerous investigators in a variety of cancers, with the finding that those with the highest levels of HIF-1 alpha in primary tumor biopsies are most likely to die of their particularly cancer. There is an association between expression of HIF-1 alpha and increased patient mortality in several cancers, he notes, including uterine/cervical, colon, lung, pancreatic, prostate and bladder cancers.
The research team, led by Semenza, the C. Michael Armstrong Professor of Medicine and a member of the McKusick-Nathans Institute of Genetic Medicine, has worked with HIF-1 before in examining how breast cancer tumors spread from the breast to the lungs. In a previous study, the team found that HIF-1 interference in the mice with the breast cancer tumors reduced growth of primary tumors and also prevented metastasis through blood vessels leading to the lungs.
Building off of that discovery, Semenza and his colleagues injected mice with human breast cancer cells that had been genetically engineered to knock down HIF-1 protein levels. After 24 days, the mice were examined and it was discovered that compared to mice with unaffected HIF-1 levels, the mice with knocked-down HIF-1 had lymph nodes with 76 percent fewer human breast cancer cells.
"We've known that increased levels of HIF-1 are associated with increased tumor vessels and with patient mortality," Semenza said in a press release. "Now we've found that HIF-1 activity is directly responsible for the spread of breast cancer to the lymph vessels. "
The team's next step was to starve human breast cancer cells of oxygen to see which genes would respond to HIF-1, and they found that the platelet-derived growth factor B gene (PDGF-B) was five times more active when oxygen was scarce. PDGF-B increases the delivery of oxygen to cells that are not currently getting enough oxygen by stimulating blood vessel growth, and also enables individual cells to survive in low-oxygen conditions. Closer examination showed that the DNA sequence surrounding the PDGF-B gene presented regions of DNA known to be recognized and bound by the HIF-1 protein, and further tests demonstrated that the HIF-1 protein binds to the PDGF-B gene and activates it. In addition, they discovered that PDGF-B produced by breast cancer cells is pumped out of the cells and stimulates the growth of lymph vessels.
Semenza and his team treated the cancer model mice with either digoxin, which blocks HIF-1 activity, or imatinib, a cancer drug. These treatments were found to reduce tumor size by 78 percent and lymph node metastasis by 94 percent.
The two ways that breast cancers spread are by getting into lymph vessels and then spreading to the lymph nodes, or by getting into blood vessels and then spreading throughout the body, says Semenza. And women who had metastases all over their bodes, he notes, always present with lymph node metastases.
"So we were focused particularly on trying to understand how hypoxia within the cancer might promote the metastasis of breast cancer cells to the lymph nodes," says Semenza, "and what we found was that the expression of PDGF-B was increased by hypoxia, that it was dependent on HIF1, and what PDGF-B did was that it stimulated the endothelial cells that make up the lymphatic vessels. It stimulated them to divide, so there's more lymphatic vessels around in the presence of PDGF-B. And the other thing is that the PDGF-B can actually attract the lymphatic endothelial cells, so in other words, the lymphatic endothelial cells will move towards the tumor, and the lymph vessels will come into the tumor, and that will again bring them in proximity of the cancer cells. The cancer cells can then invade into the vessels and spread to the lymph nodes."
Moving forward, Semenza says he and his team will be studying HIF-1 expression in triple negative breast cancer, which is characterized by a lack of expression in three receptors: the estrogen receptor, the progesterone receptor and the HER2 neu receptor. As a result, these cancers cannot be treated by anti-estrogen therapies or the popular drug Herceptin, and instead are treated with cytotoxic chemotherapy. Unfortunately, as Semenza notes, "they tend to relapse very early after treatment and to do very poorly, so there really isn't good therapy for this group of patients at the moment. We're interested in testing whether inhibitors of HIF-1 might be useful as part of their therapeutic regimen."
Oncologists at Hopkins will be initiating a clinical study later this year to treat women with breast cancer with digoxin to see if the drug can inhibit HIF-1 activity in breast cancer patients the way it did in the animal models.
"This first trial is just a very simple trial to ask whether at the doses that can be used in patients, whether digoxin is an effective inhibitor the way it is in mice," says Semenza. "And then if we have evidence that it's working to block HIF-1, then the next step would be to perform a trial probably in combination with other cancer drugs to see if there is an actual therapeutic effect."
Funding for the study came from grants from the American Cancer Society and the National Cancer Institute, as well as from funds from the Johns Hopkins Institute for Cell Engineering. The paper's authors include, in addition to Semenza, Luana Schito, Sergio Rey, Huafeng Zhang and Carment Chak-Lui Wong of Johns Hopkins; Marco Tafani and Matteo Russo of Sapienza University of Rome; and Andrea Russo of Istituti Fisioterapici Ospitalieri.