Current Research Updates
University of Miami / Miller School of Medicine
Department of Neurological Surgery
Neuroblastoma Research Lab
August 2011
By Dr. Regina Graham
We recently presented our findings
on the effects of 2-deoxy glucose (2-DG) on neuroblastoma cell viability at the
AACR 102nd Annual meeting 2011. More and more research today is focused
on understanding tumor cell metabolism and finding novel potential targets. It
is now recognized that the cancer cell’s reprogramming of energy metabolism is
an important step in tumor development. In the landmark paper D. Hanahan, &
R. A. Weinberg: (2000) The Hallmarks of Cancer, Cell, Vol. 100, pp. 57-70,
Hanahan and Weinberg described six fundamental attributes, which drive normal
cells to become cancerous. These include
· an
insensitivity to growth arrest signals
· self-sufficiency
of growth signals
· limitless
proliferative potential
· liability
to avoid apoptosis (cell death)
· ability
to create their own blood supply by inducing the formation of blood vessels
· ability
to invade and metastasize
Hanahan and Weinberg recently added
(2011), along with the cancer cells ability to avoid immune surveillance, the
reprogramming of cellular metabolism from one using oxygen (oxidative
phosphorylation) to one using glucose (glycolysis) in order to better support
the high replicative potential of cancer cells. Many talks at the meeting were
centered on dissecting the molecular pathways involved in tumor cell
metabolism and finding points for therapeutic intervention. The reliance on
glucose may have developed when the growing tumor outgrew its blood supply and
as such its oxygen supply. Cells using glucose were able to survive this hypoxic
microenvironment. Glucose not only provides ATP (energy) for the tumor cells
but carbon, and hydrogen which are necessary components along with nitrogen
derived from glutamine for the production of DNA, RNA and proteins. In addition
glycolysis leads to the production of lactic acid, which is excreted from the
cell and acidifies the extracellular microenvironment leading to increased drug
resistance and enhanced cancer cell invasion. We have found that 2-deoxy glucose induces neuroblastoma
cell cycle arrest and cell death. Neuroblastoma cells take up 2-DG but are
unable to fully metabolize it. Combining 2-DG with inhibitors of AKT can
potentiate neuroblastoma cell death. AKT is a protein kinase plays a key role
in multiple cellular processes including glucose metabolism as well as cell
proliferation, and the avoidance of apoptosis. We found that neuroblastoma
cells increase the activity of AKT in response to 2-DG treatment and inhibiting
AKT activity increases 2-DG-induced neuroblastoma cell death by an additional
50%. AKT inhibitors have shown promise for neuroblastoma treatment and are
currently in clinical trials.
We
have recently partnered with Antonello Podda, M.D., Director of Pediatric
Neuro-Oncology and Assistant Professor of Pediatrics here at the University of
Miami and Guillermo R. DeAngulo, M.D., Hematologist-Oncologist at Miami
Children’s Hospital and are currently discussing potential clinical trials for
neuroblastoma. We are thrilled and honored to have them on our Medical Advisory
Board for the Mystic Force Foundation and the fight against neuroblastoma. We
are currently determining which novel drug would be best for a neuroblastoma
clinical trial to be conducted here in Miami.
We
would like to congratulate our research assistant Constantinos Barth who was
recently accepted to Nova Southeatern University and will be starting a masters
program in biomedical sciences from which he will transition into their medical
school. We wish Constantinos all the best. Again we thank Dr. Keith Webster and
his lab members as well as Dr. John Thompson for all their help and support.
October 2010
By, Dr. Regina Graham
Treatment for high-risk neuroblastoma remains a major
clinical problem. Despite aggressive multimodal therapy including multiple
rounds of chemotherapy, surgery and radiation, the prognosis for these children
remains very poor. We continue our efforts to find effective, less toxic
therapies for the treatment of neuroblastoma.
We have presented our research on cepharanthine at both
local meetings at the University of Miami, Miller School of Medicine as well as
internationally at the Advances in Neuroblastoma Research in Stockholm, Sweden.
At each of these venues our research was well received. We hope to publish our
findings in scientific journals very soon. We have found that clinically achievable concentrations of
cepharanthine enhanced the toxicity of several commonly used chemotherapy
agents by reversing a cancer survival mechanism called multidrug resistance. Multidrug
resistance refers to the ability of cancer cells to develop resistance to a
wide range of structurally and functionally unrelated chemotherapeutic drugs
and has been a major obstacle to the success of cancer chemotherapy. To better
understand the mechanisms of multidrug resistance in patients, we are
generating multidrug resistant neuroblastoma cell lines in the lab.
Understanding how the tumor cells develop resistance will allow for the finding
of additional therapeutic targets.
Another way to attack neuroblastoma cells is to target their
metabolism. We are currently investigating the potential of the glucose analog,
2-deoxyglucose. Cancer cells have higher rates of glycolysis than normal cells.
This observation was made by Otto Warburg who won the Nobel Prize in 1931 and
is known as the Warburg effect. The high rate of glucose metabolism
(glycolysis) by cancer cells is the basis for Positron Emission Tomography (PET) scan which uses a radiolabeled
glucose analog to image tumor burden in patients. This is also the basis for
the development of 2-deoxyglucose as a cancer therapy. Tumors take up the
2-deoxyglucose but are unable to metabolize it to get the energy needed to
grow. We are investigating ways to potentiate the effects of 2-deoxyglucose in
neuroblastoma. By understanding the cell signaling pathways induced by
2-deoxyglucose treatment, we can identify therapeutic targets. We have found
that cells exposed to 2-deoxyglucose upregulate several survival pathways and
by selectively inhibiting those pathways we can significantly increase
neuroblastoma cell death.
We are happy to announce that Constantinos Barth will be
joining the Neuroblastoma Research Group full time. Constantinos volunteered in
the lab this past summer and became quite skilled in the techniques commonly
used in the laboratory.
Finally, we would like to thank Dr. Keith Webster for
allowing us to continue to use space and equipment in his laboratory as well as
all the members of the Webster lab. We also thank Dr. John Thompson for his
continued intellectual input and technical support.
January 30, 2010
By Dr. Regina Graham
The primary goal of this research group is to find novel and effective treatments for neuroblastoma. We have a two-pronged approach to address this goal. One approach is to understand the molecular signaling pathways activated in neuroblastoma, which will allow the identification of possible therapeutic targets. The second approach is to address the immediate need for more effective neuroblastoma treatments. To this end we have amassed over 75 different drugs or reagents to test in our laboratory. These drugs include traditional chemotherapy agents used for neuroblastoma, as well as drugs developed for other diseases. In addition we are evaluating the potential of herbal medicines to supplement or potentiate the cytotoxic effect of traditional chemotherapy regimens.
The investigation of “off-label” use of various drugs in the treatment of cancer is not new. We are currently evaluating the potential of drugs approved for other cancers as a neuroblastoma treatment. For example, bone and bone marrow are common sites of metastasis in neuroblastoma; therefore, we are currently examining some of the drugs that are approved for multiple myeloma, a cancer of the bone marrow plasma cells. Besides similarity in the location, both myeloma and neuroblastoma cells can activate osteoclasts which can lead to bone lesions. Another off-label drug we have looked at are the proton pump inhibitors. Proton pump inhibitors are often used to reduce gastric acid production and are prescribed for gastroesophageal reflux disease (heartburn) and ulcers. However, these proton pump inhibitors can also inhibit a different type of proton pump, the vacuolar ATPase (V-ATPase). The V-ATPase is often upregulated in cancer cells and contributes to the maintenance of a neutral intracellular pH, especially in solid tumors. Inhibiting the V-ATPase can decrease intracellular pH and induce cancer cell death.
Cepharanthine, which is isolated from the plant Stephania Cepharantha Hayata, has been widely used in Japan to treat both chronic and acute diseases for years without serious side effects. We are currently investigating this plant extract as a neuroblastoma treatment. We have found that at higher concentrations, cepharanthine induces neuroblastoma cell death, and at lower concentrations cepharanthine sensitizes neuroblastoma cell lines to vincristine and other chemotherapeutic agents by reversing multi-drug resistance. We are presenting these findings at the American Association of Cancer Research (AACR) annual meeting taking place in Washington DC in April 2010. Researchers around the world attend this meeting to learn about the latest breakthroughs in cancer research.
We now have six established neuroblastoma cell lines as well as a primary neuroblastoma cell line derived from a bone marrow aspirate of a neuroblastoma patient. We use these cell lines for our drug screening assays. In addition we have several non-neuroblastoma cancer cell lines, as well as non-cancerous cell lines used for comparison. The ultimate goal of our drug testing is to evaluate the potential neuroblastoma treatment in an animal model. One of the neuroblastoma cell lines expresses firefly luciferase, which can be used for metastasis studies. Using the firefly-luciferase expressing neuroblastoma cell lines in combination with non-invasive bioluminescent imaging we will be able to monitor neuroblastoma tumor growth and metastasis. We are currently setting up different metastasis models with these cells. In one model, the luciferase-expressing cells are injected directly in the bone marrow, the most common site of neuroblastoma metastasis. In the other models the cells are injected into the vasculature via the mouse-tail vein or into the heart. In these models the cells travel throughout the body and metastatic tumors develop at multiple locations. Lastly, we have successfully grown primary neuroblastoma cells derived from bone marrow aspirates in both NOD/Scid mice and the less immunocompromised Nude mice. We are currently planning experiments to test cepharanthine and other reagents that have shown promise in our cell culture experiments, in animal models.
Finally, we would like to express our continued gratitude to Dr. Keith Webster for providing laboratory space and use of the hypoxia chambers, and to Dr. Billy Thompson who regularly contributes to animal experiments and general technical support. We would also like to thank Dr. Sarah Woodrow who helped carry out the injections of neuroblastoma cells into the bone marrow.
August 2009
By, Dr. James Guest
Since the last research report there have been several developments that have established our research efforts more effectively.
Firstly, Dr. Regina Graham, Ph.D was appointed as an assistant professor to the Department of Neurological Surgery at The University of Miami. Currently , Dr. Graham is working full-time on neuroblastoma projects. Dr. Graham has a strong background in cancer research and is a wonderful asset to our team.
We have purchased mice of a highly immune compromised strain for tumor testing and some animals have been injected with tumor cells.
For most tumor testing in animal models, the tumor injections are made under the skin of the flank. This is a preferred method because it is easy to monitor the growth and size of the tumors visibly and by palpation. However, it is not a very relevant location for neuroblastoma which usually starts within the body near the spine and then metastasizes to the bone marrow. It is the tumor in the bone marrow which is especially difficult to eradicate and we would like to be able test our therapies in an animal model of bone marrow neuroblastoma.
We have entered into several agreements and collaborations to share molecules for testing, covering a wide spectrum of drug mechanisms. Several drugs have been tested in the cultures of neuroblastoma. Some of these have shown potent cell killing.
A key issue to understand is that there must be a good correlation between the drug concentration tested in the cell culture and the drug concentration that can be obtained in a patient. Typically drug levels are limited by side effects. So, while it may be possible to kill cells “in a dish”, at a certain dose or concentration it may not be possible to achieve that same concentration in a patient without harmful toxicity. In that case the drug may be ineffective.
One way to get around this problem is that, sometimes, if you combine drugs, they will be effective at lower concentrations. Drug combinations have also been a cornerstone of cancer therapy because the combination can target multiple mechanisms promoting cell death. This also helps reduce the emergence of resistance.
The scientific method to conduct these studies is to work out a concentration- cell killing curve for a drug by itself with e.g. a line of neuroblastoma cells. Once this is known for the drugs independently, then they can be combined to see if more effective tumor cell killing occurs with lower concentrations.
In this way various drugs can be screened against a tumor cell line and the most promising are then tested in animals which bear tumors from the same cell line. Because it is much more costly to conduct the animal testing it is important to pare down the list of test agents to those which appear most promising.
The animal testing also must be conducted to show a strong effect. Ideally the drug(s) will eliminate an established tumor of considerable size, or at multiple sites. While a size reduction in the tumor is encouraging, this does not predict a true remission.
A separate important issue in the treatment of neuroblastoma concerns the role of “cancer stem cells”. Such cells have been identified in several types of human cancer. Stem cells have several survival mechanisms that allow them to survive within the human body under conditions that are lethal to other cells. If a stem cell becomes altered so that its growth is uncontrolled, it may become a cancer stem cell. It appears that if these cells are not killed tumors will recur. We are trying to understand why bone marrow may harbor neuroblastoma stem cells so effectively. As mentioned in previous reports we think the low oxygen tension of the marrow makes the cells more resistant to killing. Another possibility is that the neuroblastoma stem cells receive signals from the bone marrow cells that robustly promote their survival.
February 2009
By, Dr. James Guest
The core of our research endeavor is to grow neuroblastoma cells in culture and transplant them into mice to form tumors. Both steps are necessary in order to test treatments. By having tumors growing in mice, and also by cryopreserving them, we are able to start the experiments over if we lose the cell cultures, which is always a risk. Because our cultures are long term, and in a specialized chamber, we are constantly battling with culture infections.
We have now grown neuroblastoma cells from 10 bone marrow specimens. There is an important difference between the early and later specimens. The most recent specimens are less differentiated. They grow under conditions that are used to cultivate cancer stem cells, and we are testing for markers of “stemness”, such as CD 133. We believe there is significant value in testing Sal's and other childrens cells. Because they are specific to each childs genetic makeup, the tumor cells may contain a unique pattern of mutations that affect their growth and response to different types of therapy.
We've included two established cell lines we have recently received from the University of Vermont and will soon begin including tumor cells from other children currently in treatment.
Transplants of bone marrow 5 have consistently yielded tumors in nude mice, some of which have been metastatic. This provides a good model of Sal’s disease. We have been able to recover these tumors back to cell culture and regrow them. Following transplant of several animals with bone marrow 8, tumors formed rapidly, being visible at 8 days. Then, unexpectedly they regressed. We are waiting to see if the tumors grow again. Among the reasons we have considered for this regression are that the tumor was rejected by the immune system. This is unexpected in nude mice which lack several immune system components. Another possible cause is that the extremely rapid growth outstripped the blood supply, and caused the tumors to die from lack of nutrients. It is always important to pay attention to the unexpected in science, since such observations may lead the way to important new knowledge.
We also submitted a letter of intent to a cancer foundation to request funding. The research work is time consuming and the materials are quite expensive. Sustaining the research effort in the long run is likely to require several sources of support . Dr. Gina Graham, PhD is now working full time on the project. I continue to work on the project about 12-15 hours per week and Dr. Billy Thompson, PhD also dedicates time to this effort. Dr.Keith Webster, Professor of Cell and Molecular pharmacology continues to permit our use of the hypoxia chamber which is a key aspect of deriving and testing cell cultures. We would benefit from a skilled technician with strong experience in cell culture and molecular biology techniques.
Recently, Gina began an extensive series of drug tests using assays that measure cell death and change in rate of cell division. She selected several currently used cancer drugs. Several of these showed a minimal or no significant effect on cells under the testing conditions. However, a drug called MG-132 was able to kill a very large percentage of these cells in 48-72 hours. Gina confirmed that the death mechanism involved “apoptosis” and we visualized numerous cells under the microscope showing apoptotic cell death. Mg-132 is a proteasome inhibitor. This means it blocks the protein complex within the cell that breaks down “used” proteins. Inhibition of this complex leads to accumulation of some proteins such as p53 that promote cell death. MG 132 is not approved for use in humans, although a similar drug called Velcade is approved for multiple myeloma. We have performed preliminary tests with Velcade at different doses on the cell cultures, and at doses of 1 micromolar the cells are heavily killed. Gina also tested drugs that function by some of the alternate mechanisms attributed to MG-132 and Velcade such as calpain inhibition, but these drugs did not have substantial effects.
The cell killing by Velcade and MG-132 is very impressive, however, it is essential to verify that the drugs will work to kill neuroblastoma cancers in mice. We will study this over the next several weeks.
Once you start growing cancers in mice and then recovering them to cell culture and retransplanting it is necessary to confirm that you are still studying a human-derived neuroblastoma as there is a small chance of causing a mouse tumor. For this reason we are having BM 5 cells tested for the presence of the Y chromosome, which should be absent in the mouse cells since they are female.
These results with proteosome inhibitors are very encouraging. To cure these children, it may be necessary to use a combination of drugs, or other treatment methods since it is possible their cells could develop resistance to the proteosome inhibitors. We are very interested to see if these cells contain cancer stem cells. There is some evidence that such stem cells underlie several forms of human cancer, and that they may be highly resistant to conventional therapies.
By, Dr. James Guest
December 6, 2008
Regina Graham will be joining us at a 50% time commitment. She will be supported by the Mystic Force Foundation with an appointment in Neurological Surgery at the University of Miami. She is a PhD with extensive post-doctoral experience in molecular biology and cancer biology. Recently she has been studying breast cancer using a flourescent luciferase model which allows imaging of human-derived tumors living inside mice. She has been performing this work under the supervision of Dr. Keith Webster, a well-known scientist in the Department of Cellular & Molecular Biology at the University of Miami. We hope to adapt this techinique to neuroblastoma studies.
Over the past few weeks we have successfuly grown tumors in mice using a bone marrow derived from the fifth marrow biopsy. The tumors have appeared as metastases in lymph nodes and the spleen. Although the cells appeared highly undifferentiated, when recovered to cell culture, they showed evidence of neuron ganglion cell differentiation. This is encouraging and indicates that they have retained the potential to differentiate. We will test if the cells further differentiate in response to retinoic acid, an important drug used to stabilize neuroblastoma minimal residual disease. It is thought that differentiated neuron cells are less aggressive and divide rarely.
We are also starting to test the bone marrow-derived cell lines for markers that stem cells are present. This will help us determine if a sub population of tumor cells have properties that may allow them to evade many current therapies.
This recent work has been conducted jointly between Dr. Webster and Dr. Guest's labs. Some diagnostic studies were provided free of charge by the UM Department of Pathology. Assistance of Dr. Billy Thompson is deeply appreciated.
October 2008
By, Dr. Steven Vanni; Neurosurgeon, Researcher & Sal's Dad
It has been one year since Salvatore was first diagnosed with Neuroblastoma. Being a neurosurgeon this was a diagnoses I never wanted to hear. I have had patients in the past with this disease and their outcomes, to say the least, were not good. I felt we needed to do something more.
When our research started we first focused on evaluating the therapeutic effects of the various treatments on Salvatore's bone marrow. As simple as this sounds, it takes hours a day to extract the cells from the bone marrow. The Neuroblastoma cells are separated from the normal cells and then under a microscope we evaluate the cancer cells to see which are dying & which are growing. We try to characterize the response to figure out the effects of the various agents he's been exposed to. One of the first things we learned is that these cells don't grow well in a normal oxygen environment. Just to keep the cells alive we had to borrow a special chamber to keep the oxygen at at a lower than normal level. This was a very interesting fact that led us to the conclusion that one way to perhaps kill these cells is to figure out how to increase the oxygen concentration they are exposed to. This low level of oxygen unfortunately can lead to the cells being infected by bacteria that tend to flourish in this environment. This has caused us to have to repeat these experiments on numerous occasions. To date all of this has been done in house, by Dr. Jim Guest, my friend and colleague who has donated all of his time to this. We initially had employed a research fellow, Nicholas, who worked very hard on this but, due to visa issues, which are a nightmare since 911 had to return back to South America. Currently we are injecting Sal's cells into mice to try to reproduce the cancer to establish a working model of his disease. From there we can test various therapeutic agents in a living model. We are looking into purchasing a new chamber to eliminate the infection problems we've been experiencing. But even something as simple as this, that doesn't allow in outside air and can maintain an environment of low oxygen costs thousands of dollars. We need to hire a full time research fellow whose sole purpose is to test various therapeutic agents on Sal's as well as other childrens cancer cells to develop less toxic treatments for our children. Also to implement the studies we think are promising, to analyze the data from these experiments and then to publish the information so it can be shared with other researchers. These endeavors are not without significant cost and funds are needed to employ the help we desperately need.
Countless hours are spent reviewing abstracts and papers that have shown some promise and analyzing the data to understand the mechanism which the therapeutic intervention may have shown benefit.
We have contacted many of these researchers but most tell a very similar story, it is too hard to get government funding, and drug companies aren't interested in curing 600 kids a year, it is not profitable for them. A sad but realistic truth. But there are ways around this, that's were we can all make a difference. We have been asking for proposals from other researches both here and abroad whose early work has shown great promise. By funding their studies and guiding their research we can gather crucial information to further the fight. This can help us set up treatment protocols to help our children today.
Some of this research can be done in house at the University of Miami, but a lot of it needs to be spread out at various institutions. Why should we fund this, you may ask, versus a drug company. Our goal is not to make a profit. It is saving the lives of these innocent children.
A recent proposal which we are reviewing, is to further a study that was recently published that uses a polio virus that has been altered to infect neuroblastoma cells and may be able to kill them. You may be wondering, why a polio vaccine, interestingly polio attacks nerve cells. Neuroblastoma is a cancer that grows from nerve cells, so by being able to infect nerve cells but only selectively killing the abnormal ones, is a unique and promising concept. Early studies have shown a surprising response, but little available funding has halted this project, we would like to get it restarted. What does it take? Money, the 2 year cost just to further the testing in mice models is about 80,000 dollars a year. This is just one example. This doesn't include toxicology testing and the countless other studies needed to bring this to clinical use. Antibody treatments are also very promising similar to one that Sal is currently receiving at Sloan Kettering in New York. But these are made from mice which can only be used for a short time until the child becomes immune to it due to the portions made from mice. The estimated cost to make a humanized version is approximately 2 million dollars. It doesn't make financial sense for a drug company to make it.
We can make a difference, from the generous support by people like you. We can gain crucial information that can help us cure this deadly disease. We can support institutions which are showing promising results. We can initialize studies we think are promising and do these at the University of Miami.
Please help us help our children.
Mystic Force Foundation is a 501(c)(3) tax exempt organization
