A WOUND THAT NEVER HEALS
Research is revealing the intimate links between inflammation and malignancy; the work has important implications for cancer treatment and prevention
BY DARRELL E. WARD
A cut on the finger oozes blood on the outside; inside, it initiates an acute inflammation response. Macrophages move to the injured area and release chemical messages — cytokines and chemokines — such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), platelet-activating factor and prostaglandin.
The macrophages arrive in M1 mode, killing invading microbes, mopping up cell debris and eliminating dead tissue. They are aided by incoming neutrophils.
As the inflammation response progresses, the area around the cut reddens, swells, feels warm and throbs with pain. And the role of the macrophages changes. They shift to M2 mode. They release cytokines that promote tissue repair, fibroblast proliferation, collagen synthesis and vascular growth. As the wound heals, the macrophages and other immune cells dissipate, and the tissue microenvironment returns to normal.
Inflammation is the immune system’s quick response to control infections, eliminate toxins and repair tissue damage. It is essential for life. Chronic inflammation, in contrast, can drive cancer development.
For example, infection by the bacterium Helicobacter pylori in the stomach or by the hepatitis B virus in the liver can create chronic inflammation that leads to gastric cancer and hepatocellular carcinoma, respectively. Long-term inflammation caused by inflammatory bowel disease (IBD) or pancreatitis also can drive cancer development.
In addition, tumor development drives inflammation. Macrophages and other immune cells regularly infiltrate tumors where, instead of attacking the tumor, they often release substances that promote tumor growth, invasiveness and metastasis. This relationship between tumor development and inflammation is so typical that cancer is sometimes described as a wound that never heals.
Researchers at The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC – James) are investigating both inflammation-driven cancer and cancer-driven inflammation.
By teasing apart the subtle interactions and intricate chemical conversations that occur between cancer cells, immune cells and the normal structural (stromal) cells within the tumors, they are learning how inflammation promotes tumor growth and then using that knowledge to develop novel, rational strategies for treating and preventing cancer.
DNA Repair and Cancer Risk
It is well known that IBD increases the risk of colorectal cancer (CRC), and that the risk increases with the duration of inflammation. The risk of CRC for people with ulcerative colitis, for example, increases 0.5 percent yearly with a cumulative risk at 30 years of 17.8 percent.
As a clinician, Kenechi Ebede, MD, saw what patients with IBD endure for cancer surveillance, including annual colonoscopies with multiple biopsies from each part of the colonic tract. It was one of the things that drew him to earn a research degree and to work as a postdoctoral researcher in the lab of Joanna Groden, PhD, professor of Medicine and member of the OSUCCC – James Molecular Biology and Cancer Genetics Program.
After 10 years, people with ulcerative colitis are often asked to consider a prophylactic colectomy, Ebede says. “But there might be people who can perhaps wait 20 years, or some who might never need the colon removed. We need to identify markers that will enable us to stratify patients according to risk to improve treatment.”
In 2011, Ebede received a postdoctoral fellowship from Pelotonia, an annual grassroots bicycle tour that raises millions of dollars for cancer research at the OSUCCC – James, and began work. “We want to identify factors that modify cancer risk in IBD and understand the mechanisms of tumor development and progression, and eventually learn how to better treat these cancers,” Groden says.
Groden and Ebede are using a mouse model of IBD to investigate whether increasing or decreasing DNA-repair capacity affects tumor development. “Funding from Pelotonia helped support the initial studies, and we were surprised by our preliminary results,” Groden says.
“This is a way to understand DNA repair and the genetic changes that can lead to colorectal cancer during chronic inflammation,” she adds. “We want to understand how some people with IBD might be more susceptible to developing cancer if they have a DNA-repair deficiency.”
The researchers are either knocking out or adding copies of a gene involved in DNA repair and replication called BLM. The mutation of BLM in humans causes Bloom syndrome, a disorder that confers a high risk of cancer.
“Without BLM, there is no DNA repair,” Groden says. “In the absence of inflammation, if we knock out BLM and induce tumors, we see more tumors. That makes sense because knocking out BLM cripples DNA repair.”
The surprise came when Ebede knocked out BLM when inflammation was present. “BLM was missing, but we got fewer tumors,” he says. “That was really interesting, and we don’t know why yet.”
When they increased BLM levels, boosting DNA repair, they got more tumors. “Our findings were very consistent but the opposite of what we expected,” Groden says. “We have hypotheses to explain these outcomes, and we’re applying for a grant to test them.
“This work will help us learn how cells acquire or protect themselves from gene mutations. It should lead to better strategies for preventing or treating tumors caused by chronic inflammation.”
Macrophages AND Metastasis
Inflammatory cells, particularly macrophages, are consistently found in the tumor microenvironment, where they influence tumor growth and metastasis, says OSUCCC – James researcher Michael Ostrowski, PhD. “But little is known about the cellular mechanisms involved.”
Ostrowski, who is chair of Molecular and Cellular Biochemistry, and a leader of the OSUCCC – James Molecular Biology and Cancer Genetics Program, is working to learn how tumor-associated macrophages promote breast-cancer invasion and metastasis.
“During normal inflammation, macrophages in the M2 remodeling phase help resolve a wound and then disperse,” Ostrowski says. “But in cancer, macrophages in the tumor microenvironment remain in the M2 phase, and they engage in chemical conversations with tumor cells, fibroblasts and epithelial cells, which promotes angiogenesis and fibrosis.” Ostrowski and his collaborators want to interrupt this cross-talk and perhaps block cancer progression and metastasis.
In a 2010 study published in the journal Cancer Research, Ostrowski and his colleagues showed that a protein called ETS2 regulates genes in tumor-associated macrophages that promote the growth of both primary tumors and lung metastases.
In addition, the researchers identified a 133-gene signature in the mouse that is also seen in human breast cancer, and found that this expression signature retrospectively predicted survival of breast-cancer patients. “More research is needed, but this could be of significant clinical importance to human disease,” Ostrowski says.
Ostrowski’s current work explores the role of ETS2 when macrophages transition from M1 to M2 phase. The transition involves a factor called CSF1, or M-CSF (macrophage colony stimulating factor). “CSF1 is important for normal macrophage differentiation and growth, but its role in tumor-associated macrophages is unclear,” he says.
Using experimental models, the researchers showed that CSF1 initiates a signaling cascade in tumor-associated macrophages that eventually activates ETS2, which then directly upregulates a set of microRNAs (miRNA).
MiRNAs are short, non-coding RNAs that regulate the translation or degradation of messenger RNA, and therefore the proteins that cells make.
“Our work suggests that CSF1 is important for the M1/M2 transition, and that turning on these miRNAs is part of that transition,” Ostrowski says.
These studies also link these miRNAs to metastasis.
“Overexpressing or removing these miRNAs raises or lowers rates of metastasis in the mouse model,” Ostrowski says.
The findings are likely relevant to humans as well. “When we compare human breast-cancer samples and lymph node metastases, we find that these miRNAs are more highly regulated in macrophages associated with lymph node metastases than with primary tumors,” he says.
Expression levels of the genes targeted by these miRNAs also correlated with patient outcome, making the findings potentially clinically important. “We are bioinformatically comparing our mouse and human signatures,” he says. “And a subset of genes regulated indirectly by these miRNAs is also present in the human samples, and they correlate with patients who don’t do as well.”
In addition, the researchers have detected macrophage-precursor cells called monocytes in patient blood samples.
The cells are in the process of differentiating into macrophages. “That doesn’t happen in healthy people,” Ostrowski says. This work is being done with OSUCCC – James researcher Jeffrey Chalmers, PhD, director of the Analytical Cytometry Shared Resource.
“These miRNAs seem to be upregulated in patients with tumors that have already metastasized to distant loci compared with patients without metastases,” Ostrowski says. “Such patients might benefit from anti-CSF1 drugs. Our work might one day enable us to stratify them into treatment groups and help us learn how well those drugs are working.”
MILKING MACROPHAGES IN THE MICROENVIRONMENT
Using microRNAs to communicate with the immune system, cancer cells can regulate the tumor microenvironment and promote tumor growth and spread.
- Cancer cell releases microvesicles that contain miRNA-21 and miR-29.
- Macrophages take up the microvesicles releasing miR-21 and miR-29 into the cytoplasm where they are taken up by endosomes.
- In the endosome, miR-21 and miR-29 bind with toll-like receptors,
ultimately causing the macrophage to release IL-6 and tumor-necrosis
- Cancer cells take up IL-6/TNF-alpha, facilitating tumor invasion and metastasis.
MiRNAs characteristically target multiple genes. MiR-155 and miR-21 target genes that encode tumor-suppressor proteins and DNA-repair proteins. Their prolonged overexpression can lead to cancer. Both are under study by OSUCCC – James investigator Carlo M. Croce, MD. “Overexpression of these miRNAs can shut down and cripple DNA repair,” Croce says.
“In healthy cells,” he adds, “if levels of miR-155 and miR-21 increase, DNA damage also increases. When this damage hits a tumor-suppressor gene or activates an oncogene, cancer can occur. It is one way chronic inflammation can lead to cancer over time, perhaps 10, 20 or 30 years.”
For example, Croce, who is professor and chair of Molecular Virology, Immunology and Medical Genetics, and director of the Human Cancer Genetics program, led a 2011 study published in the journal Proceedings of the National Academy of Sciences (PNAS) showing that inflammation stimulated a rise in levels of miR-155. That, in turn, led to a drop in levels of DNA-repair proteins, resulting in a higher rate of spontaneous gene mutations.
“That study showed that inflammation upregulated miR-155, and that overexpression of miR-155 increased the spontaneous-mutation rate, which can contribute to tumor genesis,” says first author Esmerina Tili, PhD, a researcher in Croce’s lab. “The findings also suggest that drugs designed to reduce miR-155 levels might improve the treatment of inflammation-related cancers.”
“Inflammation increases the probability of accumulating mutations that will lead to cancer,” Croce says. “Every cell has a probability of acquiring mutations. High levels of miR-155 and 21 increase that probability much more. That might explain why colorectal cancer patients who present with high miR-155 and miR-21 expression have a generally poor prognosis.
“In fact, we can say that inflammation creates genomic instability because of its action on these microRNAs. So we immediately see a connection between chronic inflammation and the induction of cancer.”
In a 2012 PNAS study, Croce and his colleagues described a novel mechanism by which miRNA promotes cancer growth and spread.
The study found that lung-cancer cells release microvesicles that contain miR-21 and miR-29, two cancer-causing miRNAs. The vesicles can fuse with macrophages and other cells in the tumor microenvironment. Inside the cells, the miRNAs bind with receptors called toll-like receptors. This, in turn, causes the host cell to release interleukin-6 and tumor necrosis factor-alpha, two proinflammatory cytokines that facilitate tumor invasion and metastasis.
“Through this mechanism, tumor cells use miRNAs to communicate with the immune system, regulate the tumor microenvironment and promote tumor growth and spread,” Croce says.
“The release of cytokines through this mechanism could enable cancer cells in metastatic sites to attract inflammatory cells and establish a nurturing microenvironment,” he notes. “This could be a crucial step in the formation of a tumor microenvironment that could potentially favor tumor growth.”
If so, targeting that mechanism might offer a new strategy for treating cancer and perhaps diseases of the immune system, he says.
Chronic Inflammation and Leukemia
A link between inflammation and hematologic malignancies is revealed by research led by OSUCCC – James researchers and co-senior authors Guido Marcucci, MD, and Michael A. Caligiuri, MD. Their preclinical study shows that high levels of the inflammatory cytokine interleukin-15 (IL-15) alone can cause large granular lymphocytic (LGL) leukemia, a rare and usually fatal form of cancer.
In addition, the researchers developed a treatment for the leukemia that showed no discernible side effects in the animal model.
“This study shows one way that inflammation can cause cancer, and we used that information to potentially cure the cancer,” says Caligiuri, who is director of Ohio State’s Comprehensive Cancer Center and CEO of The James Cancer Hospital and Solove Research Institute.
Published in the journal Cancer Cell, the study showed that exposing normal, human, large granular lymphocytes to high levels of IL-15 for prolonged periods causes cell proliferation, chromosomal instability and DNA hypermethylation.
“Normally, IL-15 stimulates the development and proliferation of natural-killer cells, innate immune cells that destroy malignant and virus-infected cells,” says first author Anjali Mishra, PhD, a postdoctoral researcher in Caligiuri’s lab. “But our study shows that excessive IL-15 activates the Myc oncogene in large granular lymphocytes.” Chronic exposure to IL-15 and overexpression of Myc, in turn, led to chromosomal instability, additional mutation and DNA hypermethylation and malignant transformation (see diagram online).
“This study showed how genetic instability and microRNAs can lead directly to cancer,” says Marcucci, who is associate director for translational research at the OSUCCC – James.
Co-author Robert Lee, PhD, professor of Pharmaceutics and Pharmaceutical Chemistry in Ohio State’s College of Pharmacy, led development of a liposomal formulation of bortezomib, a proteosome inhibitor that shuts down the cancer-causing pathway, potentially curing the malignancy in the animal model.
Leukemic mice treated with the liposomal bortezomib showed 100 percent survival at 130 days versus 100 percent mortality at 60-80 days for control animals. The researchers also found that IL-15 is overexpressed in patients with LGL leukemia, and that it causes similar cellular changes, suggesting that the treatment should also benefit people with the malignancy.
“We now plan to develop this drug for clinical use,” Marcucci says.
Such studies by OSUCCC – James researchers and their collaborators underscore the importance of treating chronic inflammation early when possible, and of vaccinating against infectious agents such as HBV that can cause it. Their work could also lead to novel treatments that delay tumor growth and spread.
Overall, research on the links between inflammation and cancer is another example of how OSUCCC – James investigators, with help from Pelotonia funding, are working to create a cancer-free world.