Tag Archives: melanoma

How zebrafish help advance cancer research

Do sharks get cancer?

Despite the widely touted myth that sharks do not develop cancer, fish of all species do occasionally develop spontaneous tumours. This is of course also true for the most common of laboratory fish, the zebrafish. In this article, I will give you a brief overview of how the unique properties of the zebrafish have been exploited by scientists to generate very useful models to study the molecular basis of various cancers.

The use of zebrafish in cancer biology goes right back to when scientists first started using them in the lab, at which point it was noticed that they spontaneously develop various kinds of tumours. However, using these naturally occurring malignancies to study cancer development is rather impractical – not only would you need a lot of fish due to the rarity of these cancers, but there would also be a lot of heterogeneity as to what kinds of tumours develop. This is clearly not ideal if you want to study the molecular basis and treatment options of one particular cancer.

From disease to model

Subsequently, carcinogenic chemicals were used to speed up the onset of cancer development. However, similar to using naturally occurring tumours, this strategy is not terribly useful for studying one particular kind of cancer, as the resulting tumours can still be very diverse(although some substances tend to always cause the same type). This approach is mostly used to identify cancer-causing chemicals during human and environmental safety testing.

To study one specific cancer type in detail, scientists started to create zebrafish carrying particular loss of function mutations (i.e. genes that lose activity due to a change), or overexpressing certain cancer-causing oncogenes (i.e. genes that cause cancer when they are overly active). Usually, this leads to the early development of only one – or at most a few – types of cancer. The first of these more specific models were acute lymphoblastic leukaemia (ALL) models, but nowadays there are models for cancers of various tissues, ranging from the brain to the pancreas.

Most of these mutant models were originally created using mutagenizing drugs followed by screening for a phenotype, but recently the research community has shifted to more targeted techniques. These make use of novel genome editing tools, such as the CRISPR-Cas9 system to switch off certain genes. The overexpression of specific genes on the other hand was usually achieved using proteins called transposases to integrate novel genetic information, but very recently the CRISPR-Cas9 system has also been tweaked to do the same.

Why study cancer in fish?

So why would anyone bother to go through this effort and do all this in fish, if we can just use the more closely related mice or rats? Apart from the lower expense and easier generation of large numbers of fish, the main reason why fish are used is that visualizing particular cells is much easier than in other organisms. This is mainly due to two factors: the existence of various transgenic fish lines in which a particular cell type is labelled, and the existence of transparent adult fish (the casper fish, as below).

Casper_fish

Transparent fish like these Casper fish shown here allow researchers to track cells inside the body of adult fish much more easily than ever before

The ease of labelling specific cell types has been exploited elegantly for studying the clonal expansion of cancer cells that drives tumor growth in vivo as it happens, as well for the study of cancer metastases. Now that adult transparent zebrafish have enabled even easier in vivo imaging, the approach has been used successfully to visualize the process by which metastases arise and cancer cells distribute throughout the body.

Understanding the origins of melanoma

A recent paper from Charles Kaufman of the Harvard Stem Cell Institute and colleagues nicely illustrates how these advantages can be very powerful indeed. In this paper published in Science magazine, the researchers used a zebrafish melanoma model that they had developed a few years earlier which expresses gene variants associated with the cancer in humans, and combined this with a newly developed transgenic zebrafish line, in which cells expressing a gene known as Crestin, which is involved in early neural development , are labelled in green. The Crestin gene is normally not expressed in adult humans, but is switched on again in melanomas. This is also why this combination is interesting; emerging melanoma cells will re-express the normally silent gene and be labelled fluorescently.

This method allowed the researchers to track melanoma development from the very first tumour cell to the macroscopically visible tumour comprised of millions of cells. The very early changes that have to occur for cancer to develop can now be studied at much greater detail than before, as these very early tumorigenic cells are extremely hard (or completely impossible) to distinguish from normal cells if they are not labelled. In this specific case the researchers identified the activation of several gene pathways that are usually involved in neural crest development in the embryo as key events in the initiation of melanoma, and believe that their findings could lead to a new genetic test for suspicious moles in patients. Their work suggests a model of cancer development where normal tissue becomes primed for cancer when oncogenes are activated and tumour suppressor genes are silenced or lost, but where cancer develops only when a cell in the tissue reverts to a more primitive, embryonic state and starts dividing.

This paper increased our understanding of the underlying biology of the very early stages of tumour development, and a detailed understanding of these early steps might be very important when developing preventative or therapeutic drugs.

Image: Kaufman, C.K., et al, 2016. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science, 351(6272), p.aad2197. DOI: 10.1126/science.aad2197

Image: Kaufman, C.K., et al, 2016. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science, 351(6272), p.aad2197. DOI: 10.1126/science.aad2197

In summary, the field of zebrafish cancer biology has made great advances in the last decade and will continue to do so with the increasing popularity of genome editing techniques. The easy visualization of particular cell types leads to distinct advantages of using zebrafish, particularly for the study of metastases and the very early stages of cancer development.

Jan Botthof

References
Kaufman, C.K., Mosimann, C., Fan, Z.P., Yang, S., Thomas, A.J., Ablain, J., Tan, J.L., Fogley, R.D., van Rooijen, E., Hagedorn, E.J. and Ciarlo, C., 2016. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science, 351(6272), p.aad2197. DOI: 10.1126/science.aad2197

Speaking out for Speaking of Research

Below is a report of a talk given by Dr. Arnold L. Goldman, a private practise vet who offered to give a talk about animal research at a school on behalf of Speaking of Research. SR regularly receives requests by students and teachers to talk to scientists, and we rely on the efforts of scientists to volunteer some of their time to give these talks. The US is a big place, and the more people offering to give talks, the better coverage we have. If you would be willing to be contacted in the future about giving a talk at a local school then please email tom@speakingofresearch.com, giving your contact details and your location.

On Thursday, April 8, 2010, the same day as the Pro-Test for Science rally at UCLA, Dr. Arnold L. Goldman, a veterinarian from Canton, CT, gave a presentation on behalf of Speaking of Research, to 75 high school seniors in North Stonington, CT. Dr. Goldman’s presentation was intended as a counterpoint to the anti-research stance of animal rights groups and was the concluding element of a senior project undertaken by senior Meredith Milligan of Wheeler High School in North Stonington.

Speaking after Ms. Stefanie Clark, a youth programs coordinator for the Humane Society of the United States (HSUS), Dr. Goldman’s presentation successfully countered HSUS arguments against biomedical research in animals. While the HSUS presentation focused on covertly obtained video footage of primates in captivity obviously intended to shock the young audience, as well as failing to distinguish product safety testing from biomedical research, Dr. Goldman presented a balanced overview.

Dr. Arnold Goldman

Using information provided by Speaking of Research, Americans for Medical Progress and the American Physiological Society, as well as his own materials, Dr. Goldman detailed the facts about biomedical research in animals. His presentation included a discussion of the moral and ethical dilemmas that exist in animal research, the actual numbers of animals used, the efforts of scientists to reduce those numbers, the myth that animal research is currently replacable, and the myth that animal data is not relevant to humans.

Dr. Goldman also went into detail about a personal experience with development of a vaccine for canine melanoma, a deadly and previously untreatable cancer, which involved one of his patients. This vaccine, originally developed using mouse DNA, eventually underwent successful clinical trials in dogs, including Dr. Goldman’s patient. The dog lived almost 2000 days beyond the expected and died from an unrelated problem. Thereafter, the vaccine’s amazing success led to clinical trials in people with melanoma, where similar success has also been achieved. The students appeared to grasp the truth that while animals used in research should be treated with respect, there is a duty to society to strive to cure disease and that these cures may help animals as well as people.

Dr. Goldman is in private practice and is also a director of Americans For Medical Progress, pro-research educational non-profit.

Speaking of Research thank Dr. Goldman for putting his time into this important cause, and urge more scientists to contact us offering to help (it is luck of the draw when we are invited to speak in schools, and where those schools will be).

RNAi: Send in the Nanobots!

The publication of the preliminary results of a small clinical trial of a new therapy called RNA interference (RNAi) online in the scientific journal Nature is causing quite a stir in the scientific community this week.  A team led by Professor Mark E. Davis at Caltech targeted the delivery of a nanoparticle only 70 nanometers in diameter containing small interfering RNA (siRNA) to cancer cells in three patients with metastatic melanoma, which reduced the levels of a protein called RRM2 that is required for the tumour growth.  This trial is the result of over a decade of research in organisms as diverse as nematode worms, mice and monkeys, but why is the result of this trial so noteworthy? And what is RNAi anyway?

Cancer genes in human melanomas have been switched off. Image courtesy of the National Cancer Institute

If you have ever studied biology you will probably be familiar with the “central dogma of molecular biology”; it describes how our genes encode the proteins that are the building blocks, and indeed the builders, of all the cells in our bodies. The very short version is that our genes are made up of sequences of double stranded DNA consisting of the deoxyribonucleotides A,C, G and T, and these sequences are transcribed by a protein called RNA polymerase into matching sequences of the single stranded messenger RNA (mRNA) , made from the ribonucleotides A, C, G and U. Another protein complex known as the ribosome then translates the mRNA sequence into a corresponding sequence of amino acids that when completed make up a brand new protein.  Our new protein almost invariable undergoes further processing but we needn’t concern ourselves with that here.  RNAi is the process where an assembly of proteins named the  RNA-induced silencing complex (RISC) binds short double stranded segments of RNA that in turn target RISC to particular mRNA sequences to which they are complementary.  RISC breaks down the mRNA molecule, preventing production of its associated protein and effectively silencing the targeted gene. The beauty of RNAi is that it allows an organism to target specific mRNA molecules for destruction, and it is a mechanism for regulating the flow of genetic information whose importance we are still only beginning to appreciate.

RNAi was discovered only 12 years ago by Andrew Fire and Craig Mello through their basic research on the regulation of gene expression in the nematode worm Caenorhabditis elegans, a discovery which earned then the Nobel Prize in 2006. C.elegans is a popular model organism for scientists studying gene function and development, its small size and simple structure make it relatively easy to follow the fate of individual cells, while as an animal it shares many of its genes and biological processes with mammals.  This turned out to be the case with when in 2001 it was shown that RNAi helps mice to control hepatitis B infection, and scientists began to examine whether RNAi could be used therapeutically (1). To do this scientists made siRNA, an artificial version of the short double stranded segments of RNA that target RISC to complementary mRNA sequences, and early experiments in mice demonstrated that siRNA induced RNAi could reduce the levels of target proteins in mice.  The first human trials of RNAi began in 2004 for the treatment of wet age related macular degeneration and at first seemed promising, but suffered a setback when further research in mice revealed that the “naked” siRNA injected into the eye in these trials actually stimulated an immune response that was responsible for at least some of the benefits seen in earlier trials (2). This was a worry as an unwanted immune response might lead to an adverse reaction if the siRNA was injected into the bloodstream rather than a small part of the eye.

In recent years scientists have been developing technologies that allow injected siRNA to evade the immune system and target only those tissues where RNAi activity is desired,  reducing the quantity of siRNA that needs to be injected and also the risk of adverse  effects due to RNAi affecting off-target tissues. Mark E Davis, a professor of chemical engineering at Caltech and one of the scientists leading these efforts, uses polymers that assemble with siRNA to form a nanoparticle that resembles a tiny ball with siRNA at its centre.  The nanoparticle shell protects the siRNA from being broken down while it is circulating in the bloodstream, and then interacts with the cell membrane to help the siRNA enter a cell so that it can do its job.  Of course he didn’t want the nanoparticle to release its siRNA payload into any old cell so he attached a protein called transferrin as a targeting ligand to the nanoparticle. Tumour cells express far more of the transferrin receptor on their surfaces than normal cells, and the hope was that the nanoparticles would bind to tumour cells in preference to normal cells.  To test whether this would work Prof. Davis team injected the nanoparticles, containing a siRNA that targeted a cancer gene, into mice that had metastatic Ewing’s sarcoma(3). They observed that the transferrin labelled nanoparticle delivered the siRNA to the tumour cells, knocked down the activity of the target cancer gene and dramatically slowed tumour growth, and when the transferring ligand was removed his effect not seen.  They also observed that the nanoparticle did not stimulate the immune system or affect any of the major organs of the mouse, indicating that their method had solved safety problems seen in earlier RNAi trials.

The targeted nanoparticle used in the study and shown in this schematic is made of a unique polymer and can make its way to human tumor cells in a dose-dependent fashion. Image courtesy of Derek Bartlett and the California Institute of Technology.

Prof. Davis and his colleagues next needed to identify an appropriate target for human trials of their nanoparticle siRNA delivery system, and decided to target the M2 subunit of Ribonuclease reductase (RRM2), a protein that is required for cell division and which has recently been the subject of a lot of research as a target for anti-cancer drugs.   They first used in vitro studies to identify a siRNA sequence that effectively targeted the RRM2 mRNA, which they named siR2B+5, and then demonstrated in mice that this siRNA could block the production of RRM2 and reduce the growth of tumours (4).  As a final safety evaluation prior to human trials they injected different doses of their nanoparticle  containing siR2B+5 and labelled with transferrin to cynomologus monkeys, whose RRM2 mRNA is targeted by siR2B+5 in exactly the same way as in humans,  and found that it was safe and did not produce any unwanted effect on the immune system (5).

The human clinical trial reported this week confirmed that transferrin-labelled nanoparticle injected into the bloodstream were safely delivered siR2B+5 to the tumours of metastatic melanoma patients, and that the siRNA knocked down the production of RRM2 protein by RNAi (6).  Of course this is only a preliminary result, at this stage we don’t know to what extent this experimental treatment will reduce tumour growth in these patients, let alone if it will cure their cancer. If it is a success it will probably need to be combined with other anti-cancer drugs to be fully effective, so it is good to know that thanks to animal research other nanotechnology based drugs such as Lipoplatin are in clinical trials that offer more potent anti-cancer activity with less toxicity than existing anti-cancer drugs. Nonetheless to focus on this uncertainty would be to miss why this small trial is causing such excitement; for the first time scientists have shown that it is possible to target RNAi therapy to a particular tissue type within the body, and that is a breakthrough that opens up a whole new area of medicine. The era of RNAi medicine has begun!

Paul Browne

1)      McCaffrey A.P., et al. “RNA interference in adult mice.” Nature Volume 418, pages 38–39 (2002) DOI: 10.1038/418038a

2)      Castnotto D. and Rossi J.R. “The promises and pitfalls of RNA-interference-based therapeutics” Nature Volume 457(7228), pages 426-433 (2009) DOI:10.1038/nature07758

3)      Hu-Lieskovan S. et al. “Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma.” Cancer Re. Volume 65 (19), Pages 8984-8992 (2005) DOI:10.1158/0008-5472.CAN-05-0565

4)      Heidel J. et al. “Potent siRNA inhibitors of ribonucleotide reductase subunit RRM2 reduce cell proliferation in vitro and in vivo” Clin. Cancer Res. Volume 13(7), Pages 2207-2215 (2007) DOI: 10.1158/1078-0432.CCR-06-2218

5)      Heidel J. et al. “Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA.” Proc.Natl Acad. Sci. USA Volume 104(14), Pages 5715-5721 (2007) DOI: 10.1073/pnas.0701458104

6)      Davis M.E. “Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles”  Nature Advance Online Publication 21 March 2010 DOI:10.1038/nature08956