Inflammation Stokes Cancer

Inflammation is our bodies’ response to infection and injury. It usually does its job and shuts itself off. But sometimes the mechanism meant to protect us can turn on us instead.

Chronic inflammation that never completely turns itself off is a hallmark of many diseases, including diabetes, infections, gastric ulcers, Crohn’s disease, and even obesity. But the effects of chronic inflammation can be even more far-reaching: A process intended to be a protective mechanism can lead to cancer. The connection between inflammation and cancer was the focus of a meeting last month in San Diego jointly sponsored by the American Chemical Society and the American Association for Cancer Research.

In inflammation, the body sends signals to immune cells such as neutrophils and macrophages, both of which are types of white blood cells, to congregate at a site of infection or injury. Once there, they release a barrage of reactive species with the goal of killing the invaders or stimulating tissue growth.

Some of the ways that inflammation can lead to cancer are straightforward. For example, various oxidative species produced during inflammation can damage DNA and lead to genetic mutations that initiate cancer. In other cases, damage to proteins or lipids interferes with their functions in ways that lead to cell death and a subsequent increase in cell proliferation. These processes are at work for decades before they manifest themselves as cancer.

“Inflammation appears to play a role at every step of the carcinogenic process,” said Peter C. Dedon, a disease researcher at Massachusetts Institute of Technology and conference chairman. In addition to being involved in the earliest stages of cancer, inflammation is also active at later stages, when it can allow mutated cells to grow better and can lead to metastasis.

Some infectious diseases have been linked to cancer. For example, chronic infection with the bacterium Helicobacter pylori—perhaps better known as the ulcer-causing bug—can sometimes lead to stomach cancer. Likewise, H. hepaticus, a liver-dwelling cousin of H. pylori, can cause inflammatory bowel disease and colon cancer, biological engineering professor James G. Fox of MIT said. These low-level, chronic infections subject the body to ongoing inflammatory responses.

The direct link between inflammation and cancer is through the various chemicals released by immune cells. One of the main jobs of neutrophils is producing hypochlorous acid—HOCl, the same compound as in bleach—and other reactive species to kill bacteria.

HOCl can trigger a number of processes involved with cancer. When it kills cells, quiescent cells start replicating to replace the dead cells, and this cell replication can result in mutations. Or it can chlorinate bases in DNA, especially cytosine, directly causing mutations that lead to cancer. Because HOCl reacts more slowly with DNA than with other biomolecules, DNA chlorination doesn’t happen often, said Christine Winterbourn, director of the free-radical research group at the University of Otago, in Christchurch, New Zealand. But if the reaction is in the right place on the DNA, it only takes a few such reactions to have a deleterious effect, she noted.

Lawrence C. Sowers, chair of the pharmacology department at the University of Texas Medical Branch, in Galveston, suspects that 5-chlorocytosine formation exerts its strongest effect through epigenetic changes rather than genetic mutations. In epigenetics, changes in gene expression result from mechanisms that don’t involve changes to the underlying DNA sequence. Epigenetic silencing of tumor suppressors happens in most major cancers, Sowers said.

The most common epigenetic change is the methylation of cytosine. This methylated DNA recruits various proteins and enzymes that block transcription. Unfortunately, these methyl-binding proteins can’t distinguish between methylcytosine and chlorocytosine.

“Chlorocytosine could mimic methylcytosine and act as a fraudulent epigenetic signal,” Sowers said. As a result, pathways that prevent tumor growth could be switched off.

Immune cells also release various reactive oxygen species that damage DNA by reacting with guanine. This reaction forms 8-oxo-7,8-dihydroguanosine, or 8-oxoG for short. Despite being one of the most common DNA lesions, 8-oxoG isn’t particularly mutagenic, said Cynthia J. Burrows, a chemistry professor at the University of Utah who studies DNA oxidation products. It correctly base-pairs with cytosine some of the time, and enzymes can repair the lesion.

But 8-oxoG has a lower redox potential than guanine, making it a hot spot for further oxidation. “When it goes one step further and is overoxidized to the hydantoin lesion, you have molecules that are very mutagenic,” Burrows said. “They never base-pair correctly with cytosine. They must be repaired, or you will certainly have a mutation.”

The primary oxidation products of 8-oxoG are spiroiminodihydantoin (spiro) and guanidinohydantoin (guanidino). In lab assays, these lesions behave similarly, Burrows said. When a polymerase manages to continue past the lesion, it invariably inserts the wrong nucleotide opposite it.

Another inflammation-related path to some of the same DNA-damage products involves carbonate radical anions. The carbonate radical, which is formed from the reaction of carbon dioxide with peroxynitrite from macrophages, can generate a guanine radical cation via a one-electron oxidation mechanism, chemist Nicholas E. Geacintov of New York University said. In addition, the radical carbonate anion can react with 8-oxoG to form spiro, or the guanine radical can react with a nearby thymine in the same strand to form a sequence-dependent cross-link. Much like the hydantoins, the guanine-thymine cross-links cause DNA polymerase to stall.

Various DNA lesions have been demonstrated in test tubes. That’s not the same as finding them in living systems.

Such lesions are now being found in animal models. For example, using liquid chromatography and mass spectrometry, Steven R. Tannenbaum of MIT, Dedon, and their coworkers have identified and measured a broad range of biomarkers of inflammation and cancer. Wenjie Ye, a postdoc with Tannenbaum, discovered in H. hepaticus-infected mice the hydantoin products Burrows had seen in lab assays. Aswin Mangerich, a postdoc with Dedon, has likewise detected 5-chlorocytosine in the same mouse model.

“This is the first time that spiro and guanidino have been measured in tissues,” Dedon told C&EN. “Now that we can detect and measure these DNA oxidation products in tissues, the next step is to determine what role, if any, they play in cancer or other diseases.”

Macromolecular damage alone is not enough to lead to cancer. “If you have DNA damage in a cell that doesn’t proliferate, cancer doesn’t result,” said Lawrence J. Marnett, a chemist involved in cancer research at Vanderbilt University.

But chronic inflammation plays a role in proliferation as well. Inflammation-related species such as nitric oxide, which is produced by macrophages, can induce cells to proliferate. They do this by activating pathways that help already-established cancer to progress and migrate.

David A. Wink, a researcher at the National Cancer Institute, has found that high concentrations of NO activate the β-catenin pathway, which is involved in cell adhesion and may play a role in cancer metastasis. Wink has also found that in a type of breast cancer known as estrogen-receptor negative, NO makes cancer cells more able to proliferate and spread.

Wink’s team has discovered that the enzyme protein phosphatase 2A (PP2A) may be a good way to target multiple NO-induced pathways for possible therapeutic intervention. This enzyme is usually turned off in tumor cells. By reactivating it, Wink and his coworkers are able to reduce levels of β-catenin and other cancer-related compounds. Working with collaborators, Wink has found two compounds—a peptide and a dithiolethione—that look like good PP2A activators and could thus be lead agents for drug discovery.

Another inflammation-induced mediator of cell proliferation that leads to cancer is the enzyme cyclooxygenase-2, Ian A. Blair of the University of Pennsylvania School of Medicine said. This enzyme’s main role is converting arachidonic acid to prostaglandins, molecules that induce even more inflammation and also lead to cell proliferation. COX-2 is upregulated in all cancers, Blair said.

But COX-2 plays a recently discovered additional role of inducing lipid peroxidation, Blair said. Some of these peroxidation products act in a feedback loop to help shut down inflammation and cell proliferation. However this potentially protective activity requires the presence of the enzyme 15-hydroxyprostaglandin dehydrogenase, which is downregulated during cancer progression, Blair said.

With these and other effects of inflammation in cancer becoming better understood, the question becomes—can we prevent the development or progression of the disease by targeting the players in inflammation?

In the case of inflammation generated in response to infectious diseases, the long-term tactic is to eliminate the bug in the first place. John D. Groopman of Johns Hopkins University has studied the role of hepatitis B virus (HBV) infection and exposure to aflatoxin (a mutagenic compound produced by molds) in liver cancer. Either risk factor increases a person’s risk of cancer—7.3-fold for the virus and 3.4-fold for aflatoxin—but the two in combination can increase the risk 60-fold, Groopman said. In a population in Qidong, China, Groopman has found that the virus and toxin working together caused many men to develop liver cancer by age 50.

Groopman believes that making HBV vaccination a public health right would help reduce such cancer risks. “We have a really good HBV vaccine,” he said. “We could eliminate this virus from the human population, but it’s going to take 100 years to do it.” In the meantime, he said, there’s a “compelling need to develop new therapeutic strategies.”

For inflammation that doesn’t stem from an infectious agent, the prevention strategy is not as clear-cut.

One tactic might be the prophylactic use of anti-inflammatory drugs, but the practice would be rife with pitfalls. First, not all inflammation is bad. Any prevention strategy that hinges on curtailing inflammation must focus on chronic inflammation while leaving the often beneficial process of acute inflammation alone. Second, many current anti-inflammatory drugs have side effects that make long-term use ­inadvisable.

COX-2 inhibitors illustrate the complexity of trying to intervene in inflammatory processes. Among the many processes that COX-2 is involved in are cell proliferation and the formation of new blood vessels. So Marnett is exploring COX-2 inhibitors as a way to slow down, or even stop, cancer progression. However, “COX-2 makes prostaglandins that at some phases of the inflammatory response stimulate inflammation, but in other phases inhibit inflammation,” Marnett said.

Tannenbaum suggests that preventing cancer by targeting inflammatory processes will involve using multiple drugs, much like what is currently done in treatment of cardiovascular disease.

But before then, we need a better understanding of the chemistry, Tannenbaum said. “Once we pin down the chemistry, we can think much better about prevention,” he said. “It’s not like finding an antibiotic to cure a bacterial disease. The inflammatory process takes 20 years or more before it causes a high risk for cancer in humans.”