Nitric oxide synthase (NOS) is a free radical signaling molecule that regulates many cellular processes, including angiogenesis, immune response, apoptosis, and synaptic communication. In the 1990s, three pharmacologists identified the signaling molecule, and the Nobel Assembly at Karolinska Institutet recognized them for the achievement, with the late Robert F. Furchgott, Ph.D., former professor of pharmacology at the State University of New York Downtown Medical Center; Louis J. Ignarro, Ph.D., professor of molecular and medical pharmacology at the University of California Los Angeles; and Ferid Murad, M.D., Ph.D., professor of biochemistry and molecular medicine at the George Washington University School of Medicine and Health Sciences, merely confirmed the molecule’s relationship to cardiovascular function. However, the scientific community acknowledged that this distinct pathway is probably related to a variety of medical conditions, from sepsis and asthma to erectile dysfunction and cancer.
Many scientists have since sought to further understand the NOS2 pathway’s role in cancer. Studies have confirmed that expression of the NOS2 gene incites an immune response and encourages malignant cells to proliferate. However, until now no researchers have tried to use drugs—nitric oxide inhibitors—to target the pathway for cancer.
Jenny C. Chang, M.D., director of Houston Methodist Hospital’s cancer center and professor of medicine at Weill Cornell Medical College of Cornell University in New York, is investigating the role NOS plays in triple-negative breast cancer. She has homed in on inducible nitric oxide synthase (iNOS), one of three enzyme isoforms of NOS that researchers have already confirmed plays a role in pro- and anti-inflammatory processes. iNOS has a closely observed relationship with the progression and metastasis of the most aggressive and treatment-resistant cancers.
The expression of iNOS increases tumor aggressiveness by regulating cancer stem cells and cells that also have the potential to metastasize. Several studies have associated iNOS expression with the worst outcomes for breast cancer, particularly estrogen receptor–negative breast cancer, in which tumors are hormonally unresponsive and not treatable by endocrine-targeted therapy such as tamoxifen and aromatase inhibitors.
“If you think about epithelial cells, they’re like cobblestones. They’re very neatly stacked,” Chang said. “These epithelial cobblestones change to spindle-like mesenchymal cells. When they’re more mesenchymal-like, they’re easier to enter into, say, blood vessels and more metastatic. It actually changes the polarity; it makes it more aggressive, more invasive.”
Chang and colleagues collected stem cells from cancer patients and suppressed about 500 genes believed to be important in triple-negative breast cancer. The researchers associated mutations in MLF2 (myeloid leukemia factor 2) and RPL39 (a ribosomal protein) genes in human patients with the worst survival rates of triple-negative breast cancer. MLF2 and RPL39 were associated with the production of NOS, which helps vascularize tumors.
Chang’s studies follow in a long line of research on this pathway. A team of molecular biologists at the National Cancer Institute have studied NOS for more than 25 years. David A. Wink Jr., Ph.D., a senior investigator in the molecular mechanisms section, and Stefan Ambs, Ph.D., M.P.H., a senior investigator in molecular epidemiology, discovered that the NOS pathway is crucial to the mesenchymal transformation in many cancers besides breast cancer: solid tumors, including lung, brain and pancreatic cancer, as well as melanomas. In a literature review, Wink observed that studies have determined that nearly all cancers have higher expression of NOS2.
“What we have found remarkably is that nitric oxide is activating all the oncogenic pathways simultaneously,” Wink said. “This is why it’s so incredible. We can show this in the petri dish; we can show this in the animal.”
However, NOS can exert both positive and negative effects. The pathway serves multiple functions in cancer physiology. In some instances, NOS levels could be responsible for tumor suppression, whereas in other cases they are related to tumor progression and metastasis.
David A. Wink Jr., Ph.D.
Chang found that several iNOS inhibitors stopped metastases and cancer stem cell renewal in triple-negative breast cancer. Overall, her preliminary research indicates that inhibiting iNOS reduced tumor growth and metastases. It was particularly effective at preventing lung metastases.
Nancy E. Davidson, M.D., directs Pennsylvania’s University of Pittsburgh Cancer Institute UPMC Cancer Centers, where she is also associate vice chancellor for cancer research and professor of medicine. She said it will be necessary to determine how an iNOS inhibitor could factor into other courses of treatment for triple-negative breast cancer, since some patients respond well to chemotherapy. “I think part of what we have to realize is that triple-negative is actually a whole family of a different kind of cancer,” she said, adding that as many as six subsets exist.
Chang has identified one promising drug that researchers have already extensively tested on heart patients. Initially, scientists developed the nitric oxide inhibitor l-N-monomethyl arginine (l-NMMA, or tilarginine), to treat patients with cardiogenic shock, which can arise with acute heart attack. Cardiogenic shock, which occurs when part of the heart muscle can no longer pump blood, may occur after coronary angioplasty but not necessarily in all heart attack patients. However, it’s the leading cause of death related to acute heart attack, and overall the condition has a fatality rate of 50%–70%.
Early investigations of the drug found that l-NMMA held promise to reverse cardiogenic shock, but a phase III randomized, double-blind, placebo-controlled trial conducted in 2005 and 2006 produced disappointing results. The researchers designed the study for 658 patients, but they stopped enrollment at 398 after a planned futility analysis. Since then the drug has sat untested and unused, Chang said.
But studies conducted on l-NMMA for cardiogenic shock show that patients tolerated the drug well and experienced few serious side effects, which is a main reason why Chang is interested in pursuing this iNOS inhibitor for a trial on triple-negative breast cancer patients.
“We know the safety profile, we know what the side effects are, it’s already been in thousands of patients,” Chang said.
Other drugs have found a dual purpose in oncology. Methotrexate, used to treat several autoimmune conditions, including rheumatoid arthritis, psoriasis, psoriatic arthritis, and lupus, is also one of the most effective chemotherapies, especially for blood cancers.
As lawmakers continue to cut federal budgets for drug research and approval, finding new purposes for drugs becomes even more essential. This is especially true for oncology, where only a fraction of therapies make it past preliminary testing. Between 2003 and 2011, just 6.7% of oncology drugs in development moved from phase I testing to U.S. Food and Drug Administration approval (Ecancermedicalscience 2014;8:442; doi:10.33332/ecancer.2014.442).
“We’re trying to now decrease a typically 15-year drug development timeline by using a drug that’s already tested in humans and has made it past phase I testing,” Chang said. “We’re crunching it into a 6-month period because we know the safety of this drug and the dose we can give and start a clinical trial.”
Chang said she hopes to start an NCI-supported clinical trial on the drug this year. Ambs said that because several researchers have now confirmed the role of this pathway, it “makes a point that a trial should be started.”
Larry Norton, M.D., deputy physician in chief for breast cancer programs at Memorial Sloan–Kettering Cancer Center in New York, called Chang’s findings a “hypothesis-generating observation” worth pursuing, though he said it’s still too early to tell whether the therapy will be effective in the long term.
“One of the advantages from a scientific perspective is that triple-negative seems to have a lot of potential targets,” he said. “In this era of evolving molecular therapeutics, sometimes the more aggressive cancers are the ones we cure quickest.”