A Problem of Disordered Development

Neuroblastoma is a notoriously heterogenous disease. A neuroendocrine tumor, it is the most common extra-cranial solid tumor in children. It is known to spontaneously regress and disappear in infants. But it can also metastasize and develop chemoresistance, in which case the options for treatment are limited, toxic, and seldom curative. Carol Thiele, Ph.D., Head of the Cell and Molecular Biology Section in CCR’s Pediatric Oncology Branch, has been studying the molecular mechanisms that determine whether neuroblastoma cells proliferate or differentiate since she joined the Pediatric Oncology Branch in 1983. Her insights have led to new therapeutic approaches and the discovery of a novel human gene that is likely to be fundamental both to tumor suppression and to normal development.

On the bulletin board next to Carol Thiele’s desk is a black and white photomicrograph with four panels labeled with the kind of adhesive lettering that was painstakingly applied to such images before the advent of computerized image processing. Each panel shows a collection of neuroblastoma cells that are growing under different conditions. Thiele will eagerly take it down to show visitors the cellular phenomenon that spurred her research career.

Mark Israel, M.D., was Head of the Molecular Genetics Section of the Pediatric Oncology Branch when Thiele was first working at the NCI as a Damon Runyon Scholar and looking for a full- time position. “Mark presented me with this phenomenon,” Carol explained. “If you look at neuroblastoma cells in culture, they are round, undifferentiated, rapidly proliferating cells. But, if you simply add retinoids (a derivative of vitamin A) to the mix, they stop dividing and start to look more neuron- like.” Neuroblastoma cells have a number of genetic alterations that render them cancerous. “That you could impose growth control and induce differentiation with retinoids suggested to me that the compound was acting to bypass or complement the defective mutations.” Thiele, a molecular biologist by training, was hooked into delving deeper into this process to understand its mechanisms.

Neuroblastoma cells labeled with green fluorescent protein, before (left panel) and after (right panel) treatment with retinoic acid. Retinoic acid treatment arrests tumor cell growth and induces differentiation.
Image shows neuroblastoma cells labeled with green fluorescent protein, before (left panel) and after (right panel) treatment with retinoic acid. Retinoic acid treatment arrests tumor cell growth and induces differentiation. (Image: C. Thiele)

The first use of vitamin A to differentiate cancer cells was pioneered in acute promyelocytic leukemia (APL) by Theodore Breitman, M.D., at the NCI in the 1980s. “They actually put retinoids into a clinical protocol to treat APL,” Thiele explained, “but the response wasn’t as good as in cell culture.” It turns out that only a subset of the APL patients—those with a particular chromosomal translocation—respond to retinoids. Once that was understood, retinoids became frontline therapy for those patients.

Meanwhile, other investigators were testing the effects of retinoids on different cancer cell lines without knowing how they might work. “It was definitely considered something of a touchy- feely area of clinical research,” recalled Thiele. Researchers have since demonstrated that retinoids bind to a family of retinoic acid receptors, which are actually nuclear binding proteins that regulate diverse transcriptional programs relating to cell growth and differentiation. It is also now known that retinoic acid (RA) is important for the development of certain neural subtypes. But many questions still remain about how retinoic acid can compensate for the genetic alterations that occur in neuroblastoma cell lines to produce differentiation and how to optimally translate the mechanisms observed in culture into tools for the clinic.

Zhijie Li, M.D., and Carol Thiele, Ph.D.
Photo shows Zhijie Li, M.D., and Carol Thiele, Ph.D. (Photo: R. Baer)

Deconstructing Vitamin A

To follow up on the effect of retinoic acid on differentiation of neuroblastoma cells, Thiele conducted collaborative studies to define the underlying mechanisms. “We were really lucky because one of our collaborators, Pat Reynolds, was able to insert retinoic acid into a neuroblastoma clinical trial that involved high-dose chemotherapy and bone marrow transplantation,” said Thiele. The patients were randomized to receive retinoic acid after the intensive treatments and the study showed that the kids who received retinoic acid had a longer event-free survival. As a result, retinoic acid is now part of the standard of care for patients with high-risk neuroblastoma. “It’s invigorating and motivating for basic scientists when they can actually see how their research can contribute to the development of a clinical trial.”

Continuing with their studies, Thiele and her colleagues found a subtlety in the effects of retinoic acid. They realized that at concentrations normally found in the body (much lower than the pharmacological doses), retinoic acid was turning on another receptor on the neuroblastoma cell surface—TrkB—which, in normal neurons, is a receptor for brain-derived neurotrophic factor (BDNF). Furthermore, researchers discovered that TrkB and BDNF were expressed in neuroblastoma tumors that had poor prognoses.

BDNF is a survival factor for neurons and when they are physically or chemically disrupted, neurons respond by turning on the TrkB receptor system. “We hypothesized that the expression of TrkB may be how the neuroblastoma fights back against chemotherapy and develops drug resistance,” said Thiele.

In 1996, Thiele and her colleagues first showed that BDNF and TrkB could affect the way the cells process cytotoxic drugs—the common chemotherapeutic agents vincristine and vinblastine. They then showed that if a neuroblastoma cell line is incubated with progressively higher levels of a cytotoxic drug, TrkB levels stayed the same but the cellular expression of BDNF increased as the cells became more drug resistant.

Our hope is this kind of strategy... will get more kids into a complete response.

The team also took cells that had either low or high expression of TrkB and incubated them with different concentrations of BDNF and then chemotherapy. They found that high levels of TrkB and low concentrations of BDNF had the same effect as low levels of TrkB and high concentrations of BDNF. In either case, drug resistance increased.

“So we knew that the TrkB receptor was attenuating the effects of chemotherapy in our cell cultures,” said Thiele. “The question was how.”

And the answer was the AKT signaling pathway, a major common denominator for survival factors like BDNF. Thiele and her Research Fellow Zhijie Li, M.D., adopted a strategy to restore chemosensitivity by targeting AKT with drugs. In a recent paper in the Journal of the National Cancer Institute, Thiele’s team has shown that neuroblastoma cells are very sensitive to an AKT inhibitor alone. But they now have data demonstrating that the combination of AKT inhibition and a standard chemotherapeutic agent is highly synergistic.

“We’re also excited by a recent phase 1 clinical trial of the AKT inhibitor perifosine in pediatric cancers,” added Thiele. Among the patients tested, there were four responders, which included two of the three neuroblastoma cases included in the trial. “So our hope is this kind of strategy—high-dose chemotherapy up front integrated with an AKT inhibitor—will get more kids into a complete response. We know that the complete responders have a much lower chance of relapsing over time.”

Thiele notes that they deliberately chose not to work on the translation of TrkB inhibitors into the clinic. “In studying the biology of neuroblastoma, I realized there were probably several growth factors that could have a similar effect on chemosensitivity. So instead of a therapy that combines multiple specific targets, I felt that the best bet was to go downstream to a common survival signaling node like AKT.” This approach, Thiele hopes, will bypass any relapse due to alterations in a related survival factor.

Hunting for Tumor Suppressors

Alfred Knudson, M.D., Ph.D., is credited with the development of the hypothesis that somatic loss of both alleles of a gene could lead to cancer. The now-famous two- hit hypothesis later merged with the concept of tumor suppressor genes when it became clear that the development of retinoblastoma was associated with mutations in both alleles of the retinoblastoma gene RB. “Less well known,” said Thiele, “is that neuroblastoma is the second cancer for which Knudson postulated a tumor suppressor.”

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