Malicious inflammation / Gary Stix

If we understand the chronic inflammation that contributes to heart disease, Alzheimer's disease and a variety of other diseases, we may find the key to deciphering the mysteries of cancer

More than 500 million years ago, a group of specialized enzymes and proteins evolved to protect our ancestors from attacks from the outside world. If it happened that a bacterium broke out and entered the body of a living being in the Precambrian era, the elements of the primitive immune system would have attacked the cause of the disturbance in a wild but coordinated attack - piercing its cell walls, spitting chemical toxins at it, or simply swallowing and digesting it whole. After eliminating the invader, the immune forces began to heal the damaged cells or, if the damage was too great, to bring about their end.

This inflammatory immune response was so effective that it was preserved throughout the long ages of evolution. We know this because studies have found that we share many immune system genes with the simple fruit fly - and the vertebrates and invertebrates split from a common ancestor more than half a billion years ago.

For many years immunological research ignored this innate immune system, because it saw it as nothing more than a bunch of thugs standing at the gate and striking their blows at anything that manages to penetrate through the smallest opening in the skin or shell of a living being. The researchers preferred to delve into the acquired, more advanced immune system, which trains antibodies and other weapons to identify an intruder and attack it with a precision that is absent from the untrained innate system.

In the past 15 years, the innate immune system has received the attention it deserves. Inflammation, the central feature of this system, has been recognized as a hidden contributor to almost every chronic disease, a list that includes, apart from obvious diseases such as rheumatoid arthritis and Crohn's disease, diabetes and depression, alongside very deadly diseases such as heart disease and stroke [see sidebar on page 65]. In the last decade, the possibility that the other great killer, cancer, also has an inflammatory background has been studied with great interest. "The connection between inflammation and cancer has moved to center stage in the research arena," says Robert A. Weinberg of the Whitehead Institute for Biomedical Research at the Massachusetts Institute of Technology (MIT), who highlighted the shift in emphasis in the new edition of his book, "The Biology of Cancer" (published by Garland Science, 2006).

The current revolution recognizes that the inflammatory immune state serves as a key mediator in the intermediate stages of cancer development. The beginning of a cancerous tumor is a series of genetic changes that stimulate a group of cells to divide more than usual and then invade neighboring tissues. Over time, some of the tumor cells may detach from it and establish new tumors (metastases) in distant sites. This picture was understood a long time ago. But biologists and immunologists who research cancer have begun to understand that the transition from a diseased tissue to a developed invasive cancer involves, in many cases, the diversion of cells, which in their normal function contribute to the healing of cuts and scratches, to the environment of the premalignant tissue. There they are "kidnapped" and become assistants in the cultivation of the cancerous tumor. As researchers describe the malignant condition: the genetic damage is the match that ignites the fire, and the ignition is the fuel that feeds it.

Today it is clear that a tumor is not just a mass of cells that have deviated from the right path; It also includes a support system, a vascular network and a microenvironment that contains a variety of immune cell types and the chemical signals that are transmitted between them. The tumor begins to be perceived as a law-breaking organ, whose existence is dedicated neither to blood flow nor to the elimination of toxins from the body, but to the service of its own purposes.

In the new view, it may not be necessary to remove every last cancer cell from the body. Instead, anti-inflammatory therapy may prevent precancerous cells from becoming cancerous, or stop an existing tumor from spreading to other areas of the body. Cancer patients may then be able to stay alive, just as AIDS patients are now living longer with the help of the new HIV drugs. "I don't believe that the goal is actually healing. There's no need for it," says Lisa M. Cosens, a cancer research biologist at the University of California, San Francisco (UCSF). "If you can control the disease and live your full natural life span, this is a tremendous victory."

Multiple lines of defense

To understand the connection between inflammation and cancer, you need to know how the body reacts to invaders - and how a normal healing process can turn into cancer when the inflammatory state lasts too long. If you stepped on a nail, a horrifying bunch of proteins and white blood cells felt to advance the surface of the bacteria that penetrated your heel. Among them are, for example, 20 proteins of the complement system, so called because it complements other defense mechanisms in the body, which spray chemicals on the invading pathogens until they explode into one big protoplasmic mush. While the complement system floods the area with mucus, cells called in the immunology books professional phagocytes, which are expert cells in literally swallowing, go into action.

Macrophages and neutrophils, which are among the phagocytic cells, are in fact, "pacmans" without table manners, who envelop the uninvited guests and digest them. Other soldiers in the attacking unit are natural killer cells, mast cells, and eosinophils. There is much more to the healing process than launching an attack against the invader. Platelets involved in coagulation migrate to the breach in the skin from an inner layer of skin rich in blood vessels. Enzymes direct the repair of the infrastructure. The extracellular - the proteinaceous substance in which the cells sit. A scar forms, the skin grows back, and the entire inflammatory process ends. But sometimes the inflammation does not go away. Any tissue (and not just skin) suffering from chronic inflammation due to the constant presence of pathogens, toxins or genetic damage, is A source of disease, from heart disease to cancer.

Vertebrates, beyond the first layer of defense, have additional weapons. The acquired system learns the particular molecular signature of the intruder and then uses it as a target to kill. The main players include B cells, which produce antibody molecules that neutralize pathogens or mark them for destruction, and T cells, which spur damaged cells to commit suicide or secrete chemicals that direct the activity of other immune players.

In recent years, more and more evidence has accumulated showing that chronic inflammation may be a very important factor in the transition of certain types of tumors from the pre-cancerous stage to the active disease stage. The suspicion that there is a link between cancer and inflammation has existed for a long time. In 1863, the renowned German pathologist Rudolf Virchow discovered white blood cells in malignant tissues. In 1978, Alberto Mentovani, from the Institute of Human Medicine and the University of Milan, noticed that cells from the innate immune system tend to cluster around certain tumors. In 1986, biologist Harold P. Dvorak of Harvard Medical School said that tumors are "wounds that never heal." However, the status quo was elsewhere. Until about ten years ago, many biologists dismissed the idea that the immune system is used not only to eliminate pathogens but also to hunt cells with cancerous potential. But a closer look at the microenvironment of cancerous tumors revealed the unexpected.

Double-edged sword

In the late 90s, Frances Blackwill of the Queen Mary Cancer Institute at the University of London was engaged in research on a certain type of cytokine, a hormone-like immune signaling molecule, called "tumor necrosis factor" (TNF) because it kills cancer cells when injected in large quantities directly into the tumor. But when TNF is present in the tumor in a small chronic amount, it acts very differently. In Blackwill's lab, they turned off the gene for creating TNF in mice so that the rodents could not produce the protein. To their surprise, the mice did not develop tumors. "The discovery immediately made us an 'undesirable entity,'" she recalls. "All the people who worked on TNF as an anti-cancer agent were shocked. The cytokine that was supposed to be used to treat cancer actually acted as an internal tumor stimulator."

The availability of knockout mice - transgenic mice that are created in the laboratory without an active gene of a certain type - on which the effects of selective turning off of genes can be tested, has helped to emphasize the link between cancer and inflammation. Cosens and her colleagues at UCSF, Douglas Haahan and Zeena Warb, reported in 1999 that transgenic mice with activated cancer-causing genes but lacking mast cells (another type of innate immune cell) developed premalignant tissue that did not progress to full-blown malignancy. In 2001, Jeffrey W. Pollard and his colleagues at the Albert Einstein College of Medicine described mice that had been genetically engineered in a way that made them prone to breast cancer tumors, but developed pre-malignant tissue that did not become malignant unless it recruited macrophages for help.

The new image did not completely turn the old one upside down. In fact, she finds that the immune system functions as a double-edged sword. The network of molecules and cells, second in complexity only to the brain, remains a paradox: sometimes it promotes cancer, and sometimes it inhibits it. Certain types of innate immune cells, such as natural killer cells, can actually protect against tumor growth. Others may foster malignancy only when the microenvironment is "tuned" to an inflammatory state. In another environment, they might eradicate it. Inflammation produces tumors in many organs, but not in all - and its relationship to cancers that form in the blood is not well characterized.

In their search for the culprits, the researchers often focused their microscopes on macrophages, which occupy a significant place among the white blood cells in the tumor's microenvironment. The macrophages are able to kill tumor cells or send T cells of the acquired immune system an alert that something has gone wrong. But the work of Pollard and other researchers showed in detail how macrophages are "re-educated" by cancer cells, making them obey their instructions. The macrophages become factories for cytokines and growth factors that nurture the developing tumor.

The transformation of the macrophages into a fifth recruit begins when the tumor cells send out help signals that attract cells that become macrophages upon reaching the tumor. Inside the tumor, the thriving cells grow so fast that they begin to die from lack of oxygen. A combination of a lack of oxygen and messages from the tumor cells initiates a process in which the macrophages that have arrived take on their evil identity as tumor promoters. Cancer biologists have given these rebels, who gather in and around the tumor, the name "tumor-associated macrophages".

Biologists have already been able to track the inflammatory link down to the level of single signaling molecules, and found there more solid evidence for a cancer link. For example, the substance called "nuclear factor kappa-B" (NF-kB) is a cluster of proteins that acts as a master switch for activating inflammatory genes and controlling cell death. The name of NF-kB went out in the world because its discoverers, who also patented it for medicinal use, are "science stars", including Nobel laureates David Baltimore and Philip A. Sharp, and because it was often the focus of litigation over patent rights worth millions of dollars.

In 2004 Yanon Ben-Naria, Eli Pikarski and their colleagues from the Hebrew University of Jerusalem reported on experiments in mice engineered to develop jaundice and later, liver cancer. When NF-kB was inhibited in the epithelial cells of the liver - through genetic modification, or through blocking the pro-inflammatory signaling molecule TNF, these mice developed precancerous tumors that did not progress to full malignancy. Blocking the binding of TNF to the receptor on premalignant liver cells using a TNF-neutralizing antibody prevents the initiation of the chain of molecular events that activates the main switch for the generation of NF-kB. The absence of NF-kB caused the precancerous liver cells to initiate apoptosis - programmed cell death. In another discovery that year, Michael Karin and his colleagues at the University of California in San Diego found that even in mice engineered to develop colitis, a disease that can lead to colon cancer, inhibiting NF-kB triggered apoptosis. The silencing of the pathway in inflammatory cells, such as macrophages, also disrupted the development of tumors.

To date, the clearest evidence for a link between cancer and inflammation is the data showing that inflammation promotes the transformation of precancerous tissue into malignant tissue in many types of cancer. But the biological response may also be involved in initiating the disease and sending metastases. Infection with Helicobacter pylori (which causes stomach ulcers) causes inflammation that greatly increases the chances of cancer in the upper digestive tract, and the virus that causes hepatitis C can also cause liver cancer, and these are just two examples. Pathogens may also produce free radicals, which can damage DNA. However, although it is possible that inflammation is involved in the cancer process from the beginning, there are few studies that have shown that inflammatory conditions do indeed change the DNA and provide the initiating spark.

The evidence for a link between an inflammatory state and the formation of metastases is stronger - and recent studies confirm the hypothesis. In the April 5, 2007 issue of the journal Nature, Karin's group reported that inflammation, and not genetic changes in cancer cells, triggered the formation of metastases in mice engineered to develop prostate cancer. The study revealed that cytokines produced by inflammatory cells near the prostate tumor caused the tumor cells to reduce the production of the protein that blocks the formation of metastases. This result, Karin says, may explain the surprising observation that an incision in a tumor, such as that made in a prostate biopsy, sometimes promotes the formation of metastases. If he is right, maybe the inflammation caused by the injury is to blame. Around the same time, Pollard's group reported in the journal Cancer Research on a study they did in mice, in which they found that macrophages accompany breast cancer cells in their migration to the blood vessels that will take them to distant sites, and along the way they constantly send chemical signals to their partners.

In studies on the connection between inflammation and cancer, the innate immune system has received most of the attention. But the acquired immune system - T cells and the antibodies produced by B cells that target certain molecules in invading cells - can also contribute to the pathology or fight it. For decades, immunotherapies designed to boost the T-cell response against cancer have been studied, although the results have often been disappointing [see box on previous page].

Furthermore, the emerging picture begins to reveal a complex communication between innate immune cells and acquired immune cells, which may play a role in the promotion of malignancy. Researchers working on a cancer vaccine will likely need to look at these interactions when designing treatments if they want to see results. One study showed that ovarian tumors produce a signaling molecule that attracts regulatory T cells, a subset of acquired immune cells responsible for silencing other T cells [see "Suppressing the Suppressors" by Lisa Melton, Scientific American Israel, April-May 2003]

Meanwhile, in a study published in 2005 in the journal "Cancer Cell", Cosnes and her colleagues at UCSF discovered that removing antibody-producing B cells from mice that had been engineered to be prone to skin cancer prevented the changes in tissue and the proliferation of blood vessels that are essential to the progression of the disease. In their normal function as pathogen fighters, antibodies produced in B cells circulate in the bloodstream and mark viruses and bacteria for destruction by innate immune cells. But in response to signals from precancerous tissue, the antibodies cause the innate system to cooperate in the development of the cancer. The question of how this process begins is an open research issue. One of the possibilities that has been raised is that the cancer cell sends a message to innate immune cells, perhaps to dendritic cells, which in response activate B cells. It is possible that "toll-collecting" type receptors are involved in the signaling, which were discovered to be central mediators in sending immune messages in the innate system.

Cancer inhibitors

The recognition that cancer is more like an organ than a mass of cells with DNA mutations in the cell nuclei may explain why some of the previous approaches to chemotherapy did not fare so well. "People took cells and then cultured them and stuck them into animals," says Pollard. "They grow as little balls. They do certain things there, but they are not complex tissues, whereas natural tumors are extremely complex tissue."

Instead of simply killing cancer cells - a goal towards which current drug and radiation treatments are aimed - it is possible to develop new approaches that will support the action of existing drugs by slowing down the inflammatory process. Without the involvement of the macrophages and other innate immune cells, the premalignant tissue will remain under control. Cancer could, in principle, become a chronic disease, similar to rheumatoid arthritis, which is also an inflammatory disease. "Remember, very few people die from primary cancer," says Raymond Dubois, academic director of the MD Andersen Cancer Institute at the University of Texas, which studies the effect of anti-inflammatory agents on cancer. "The patient almost always dies due to the spread of metastases."

A drug against chronic inflammation embodies a more tempting idea than the mass killing of malignant cells (and with them also healthy ones, with no choice) offered by the chemical treatments used today. Such a drug, alone, may be easy enough for daily use as a preventive treatment for those at high risk. Epidemiological and clinical studies have shown some success in stopping the development of certain solid tumors with the use of non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin. Research continues in the search for a more selective blockade of the production of prostaglandins, which are the regulatory molecules that NSAIDs treat. Drugs that inhibit the production of prostaglandin E₂ may stop the inflammatory and cancerous processes without side effects that cause damage to the heart, as happens with the use of drugs like Viox, and without the digestive problems caused by the NSAIDs that preceded it [see "The goal: pain", by Gary Stix, Scientific American Israel, April-May 2007] . The anti-inflammatory effects of the statins used to lower cholesterol are also being examined.

Several treatment options already exist. The drug Avastin inhibits the production of VEGF, a substance that promotes the proliferation of blood vessels, although oncologists have to deal with other molecules in the tumor's microenvironment that promote the growth of new blood vessels. Medicines developed to treat more well-known inflammatory diseases may also help in the fight against cancer - and the possibility of producing drug cocktails from them, such as those used to fight HIV and also containing blood vessel inhibitors and cell killers, is being examined.

TNF inhibitors have been approved for the treatment of rheumatoid arthritis, Crohn's disease and other diseases, and are currently in clinical research stages for the treatment of solid tumors and various types of blood cancer. The drug Rituxan, a monoclonal antibody that suppresses B cells in rheumatoid arthritis and B-cell lymphoma, may prevent the inflammatory response that feeds solid tumors. Cytokines and other molecules (IL-6, IL-8 and CCL2, among others) are also potential targets, as is NF-kB.

Some of the existing compounds, including NSAIDs and another found in the spice turmeric, have an effect, at least partially, by inhibiting NF-kB. But in the major laboratories for drug development, studies are being conducted on highly selective inhibitors of this molecular switch, and many of them target the enzymes that regulate its activity (such as I-kB kinase).

Chemical Trojan horse

One of the groups is considering a particularly ambitious treatment, like a molecular Trojan horse. Claire Lewis and Monita Mothana, from the University of Sheffield in England, and their colleagues, devised a drug delivery program that utilizes the natural attraction of macrophages to low-oxygen growth areas. They engineered macrophages that deliver a therapeutic virus to low-oxygen tumor areas that, due to the poor oxygen supply, do not respond well to conventional chemotherapy and radiation treatments. After the macrophages reach the tumor (so far only in culture in the laboratory), each of them releases thousands of copies of the virus that infect the cancer cells. A protein found in these cells activates the therapeutic gene in each virus. As a result, a cell-killing toxin is synthesized. "The macrophage migrates to the site and does what we want it to do instead of promoting tumor development, as is its custom," says Lewis.

The exact outline of an anti-inflammatory strategy for cancer treatment is still unclear. Altering the function of immune cells that serve as a protective wall against pathogens carries its own dangers. "This is a very complex issue," says Dubois. "If you miraculously turn off the immune system, you will have problems with infections that will take advantage of the situation, like with AIDS." The use of TNF blockers in other inflammatory diseases has been linked to tuberculosis and other infections, and possibly even lymphoma. Furthermore, inhibition of the NF-kB pathway can, paradoxically, promote cancer in some cases. The delay may cause tissue damage and an abnormal tissue regeneration process, resulting in cancer.

And it still seems that there is a good chance that a new generation of anti-inflammatory agents will join the array of chemotherapy weapons. Chronic diseases - and the inflammatory processes underlying them - are among the most prominent symptoms of an aging population. "We're all a little too inflammatory," explains Pollard. Treating the whispering embers surrounding the tumor, and not just the mutated cells, may turn cancer into a disease you can live with.

And more on the subject

Smoldering and Polarized Inflammation in the Initiation and Promotion of Malignant Disease. Frances Balkwill, Kellie A. Charles and Alberto Mantovani in Cancer Cell, Vol. 7, no. 3, pages 211–217; March 2005.

Distinct Role of Macrophages in Different Tumor Microenvironments. Claire E. Lewis and Jeffrey W. Pollard in cancer research, Vol. 66, no. 2, pages 605–612; January 15, 2006.