Flipping cancer's switches

Roland Rad on his PiggyBac journey to expose cancer causing genes

Genes are switches - they are the controls for our biological existence. Cancer strikes when those controls are set wrong - pushing our cells into overdrive; allowing them to duplicate uncontrolled, sometimes uncontrollably.

Dr Roland Rad, clinician scientist at the Wellcome Trust Sanger Institute, has developed a novel system - a tool he hopes will help to unravel that most compelling of questions in cancer research: which of our genetic switches, when flipped, can lead to cancer?

Roland Rad. Clinician scientist at the Wellcome Trust Sanger Institute.

Roland Rad. Clinician scientist at the Wellcome Trust Sanger Institute. [Genome Research Limited]

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Millions of years ago before humans discovered the substance called DNA, before genes were identified as the source of all our tissues, before the letters A, T, C and G had been spelt out by a human hand - the DNA that made up our ancient vertebrate ancestors was quite different from ours. Among linear stretches of DNA, were tiny snippets that could spontaneously jump around the genome. These snippets - these jumping genes - have long been inactivated in modern human genomes. They were damaging, unpredictable and selfish DNA parasites, eventually selected against by evolution.

Now, millions of years later, jumping genes have become critical in the quest to uncover the genetic basis of cancer.

Roland Rad, a training gastroenterologist, has dedicated his first three years in the Sanger Institute's Mouse Genomics team to finding new ways to harness jumping genes - or transposons - to systematically uncover genetic pathways that lead to cancer.

"There are lots of ways to go about seeking out those genes that are critical in the pathway to cancer," explains Roland. "And different methods can complement one another. What our team is working on involves using transposons to create genetic mutations that trigger the development of tumours in mice. We can then peer back into the genome searching for the molecular signature of the transposon, which will point us to specific gene that has been hit.

"This is forward genetics."

Transposons for cancer research

In 1948, five years before Watson and Crick's description of the structure of DNA became the iconic biological moment of the 20th Century, a botanist called Barbara McClintock made another groundbreaking observation. Looking in fine detail at the cellular structure of maize, she noticed something peculiar. Within the chromosomes, she saw a jumble: with sections added, removed and moved around.

What McClintock had observed, for the first time, was the effect of DNA transposons.

The effect of transposons in Maize. Botanist, Barbara McClintock, first observed the effect of DNA transposons when looking at the cellular structure of abnormally coloured kernels of maize.

The effect of transposons in Maize. Botanist, Barbara McClintock, first observed the effect of DNA transposons when looking at the cellular structure of abnormally coloured kernels of maize. [Credit: PLoS, DOI: 10.1371/journal.pbio.0040353.g001]

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"Transposons are cut and paste genes," says Roland. "We know that biological function is determined by a sequential genetic code - so you can imagine how it might wreak havoc if a section of DNA is randomly pasted into a sequence. It sounds destructive but, in experimental terms, it is a truly exciting opportunity that has revolutionised our approach to cancer research."

In the late 20th century, teams started thinking about how they might harness these wayward genes to create experimental models for research into human diseases. The first major breakthrough came in 1997 when researchers successfully activated a transposon called Sleeping Beauty, which had been dormant in the salmon genome for some 15 million years, in fish, mouse and human cells to set it in motion jumping around the genome.

For cancer geneticists who had been using the mouse as a model the possibilities were clear. If a transposon moves around the mouse genome at random, it will eventually land in a cancer causing gene; if it disrupts that gene in the right way, it will give rise to cancer. Of course, genetic similarities between human and mouse mean new insights into the development of tumours in mice would yield new insights into the development of tumours in humans - insights that could shape the drug treatments of the future.

The last decade has seen cancer geneticists around the world embark on a quest to harness the power of transposons to root out cancer genes.

In 2006, Roland Rad enrolled in that search when he joined the Sanger Institute's Mouse Genomics team, led by Professor Allan Bradley. For the past three years, he and the team have been working to fine tune their own system - the PiggyBac transposon.

A PiggyBac ride

"The transposon we are using was discovered in the moth," says Roland. "Our work has been to transform that transposon to develop a system that allows us to be as comprehensive as possible in our hunt for cancer genes. We needed a system that would work in a broad range of experimental settings, to answer a broad range of scientific questions."

Microinjection of embryonic stem cells into a 3.5 day blastocyst. Together with the Mouse Cancer Genetics team, led by Dr Pentao Liu, the team generated mice with elements of the PiggyBac system. They then bred the mice, to create the working PiggyBac model.

Microinjection of embryonic stem cells into a 3.5 day blastocyst. Together with the Mouse Cancer Genetics team, led by Dr Pentao Liu, the team generated mice with elements of the PiggyBac system. They then bred the mice, to create the working PiggyBac model. [Genome Research Limited]

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From the outset, the advantages of PiggyBac were clear: it was a new kind of transposon. Where other transposons leaves behind scars in the healthy DNA as they jumped around the genome, PiggyBac doesn't leave undesired damage when jumping out of a DNA segment..

"To start the hunt," says Roland, "we worked in collaboration with the Institute's Mouse Cancer Genetics team, led by Dr Pentao Liu, to create mice that carried a transposase - a genetic engine that can move genetic material around the genome - and mice with various transposons - specially designed cargoes that can be shipped around the genome by the transposase. By breeding these mice we were able to bring together the two main elements of transposon mutagenesis and establish the fundamentals of the PiggyBac system."

There are two main genetic failures that lead to cancer. The first occurs when the activity of a cancer gene - an oncogene - is ramped up, leading to uncontrolled cell reproduction; the second takes place when the activity of gene that suppresses the development of cancer - a tumour suppressor gene - is inhibited, preventing it from its duty as genetic law enforcer.

The transposons that the team used were designed to root out different types of cancer genes: three boost the activity of genes allowing the team to identify oncogenes and two inhibit gene activity allowing the team to identify tumour suppressor genes.

The PiggyBac transposons also behaved differently, dependent on where they were used: some had an effect only in the blood, some only in solid tissues and some had an effect in both. However, the team really wanted to develop a far more targeted approach. To achieve this, Roland and the team would need to tame PiggyBac.

Targeted treatments for solid tumours

In the late 20th century transposons were becoming a talking point in experimental genetics. Though these genes were already widely used in the study of bacteria, plants and other simple organisms, until 1997 the use of transposons in vertebrates remained unchartered territory.

Researchers had traditionally used retroviruses to disrupt the mouse genome in the search for cancer genes. And though the method had uncovered hundreds of genes behind blood and breast cancers, creating solid tumours remained beyond reach.

After Sleeping Beauty was successfully activated in the mouse genome for the first time in 1997, using transposons to root out gene targets for treatments against solid tumours had become a real possibility.

With Sleeping Beauty and, more recently, PiggyBac, fine-tuned and in a position ready to identify cancer causing genes, the door is open for a new level of molecular understanding of solid tumours and, ultimately, the development of new drug treatments.

"We have seen the success of targeted, 'intelligent' treatments in other areas of cancer treatment," explains Roland. "For instance, Gleevec, a drug targeted against mutations found in Chronic Myeloid Leukaemia, is now widely used to treat the disease. With our transposon system in place, we have the power to understand solid tumours in more detail than was previously possible.

"The future of treatment for these cancers is very much brighter with the addition of PiggyBac to our armoury of cancer research tools."

Taming PiggyBac - targeting tissues

Roland's team was keen to develop a system that could be manipulated to find genes causing cancers in specific tissues of the body - be it lung, blood, kidney or, for Roland, the gastrointestinal tract.

They would need to establish a set of controls.

"We can't just insert the transposon into the genome and let it run wild," says Roland. "Many of our mouse models would not even reach adulthood if we took this approach. We need to be able to control when and where the jumping begins."

To do this, the team use a cunning method to harness sections of sequence in the mouse genome that control the activity of genes.

"To make sure that we can control the transposon, we insert what is known as a 'stop codon' in front of the transposase," says Roland. "This is a short regulatory sequence shuts down the engine of the system. So the default position is that the transposon is dormant."

When the team want the jumping to begin, they inactivate the stop codon by inserting what is known as a 'cre-recombinase' - a special type of enzyme like a pair of scissors - that can cut out the stop codon by recognising the genetic code either side of it.

"The really clever part is that we can develop mice where the enzyme - the cre-recombinase - is only present in specific organs or tissues," explains Roland. "And that process has given us the power to control PiggyBac - to activate it only in specific regions that are of interest to us.

"It is a crucial step in the process; it allows researchers of different specialisms to answer their own burning questions about cancers of specific tissues."

Why PiggyBac?

Fine tuning PiggyBac has taken three years of hard work and collaboration from the Sanger Institute's Mouse Genomics and the Mouse Cancer Genetics teams: from developing different transposons for different screens, to genetically engineering the mice, to establishing the optimum number of copies of the transposon needed, to developing the means to mutagenise specific organs. Now, finally, the tool has been fully characterised.

"It's been painstaking at times," says Roland, "but there have been some real highs along the way: like the first time I successfully created a tumour using the model - that was a real eureka moment!"

PiggyBac now joins an expanding armoury of techniques that have been developed over the past decades to model cancer.

Fine tuning PiggyBac has taken three years of collaboration between the Institute's Mouse Genomics and Mouse Cancer Genetics teams.

Fine tuning PiggyBac has taken three years of collaboration between the Institute's Mouse Genomics and Mouse Cancer Genetics teams. [Genome Research Limited]

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Already, the team have uncovered new genes using PiggyBac. One of the genes the team exposed, called Spic, was disrupted in nine myeloid leukaemia tumours in the mice. Other genes the team identified include Hdac7, which is known to participate in the creation of white blood cells; and Bcl9, a gene whose human equivalent is thought to be involved in leukaemia.

With these findings now in the public domain, researchers around the world are free to study the genes and their human counterparts in more detail - to understand the role they play.

"Together, the genes we have found highlight how PiggyBac to complements approaches that are already out there," says Roland. "But this is just the start. With this new system, we are reaching larger and larger proportions of the mouse genome - and that can only mean more potential drug targets will be found."

There are other advantages too. PiggyBac is strong enough to carry genetic cargoes far larger than previously possible. And with room for larger genetic cargoes, the team can develop more complex genetic designs to answer more complex scientific questions.

For instance, Roland and the team were able to use the PiggyBac transposon to look at tumours in a completely new light, by inserting what is known as a 'reporter' into the genetic material carried around the genome.

"By adapting our transposon to include the reporter' we have been able to visualise the development of tumours using our model," explains Roland. "Quite literally, the added material stains the cells and as the tumour develops, the staining spreads. So we can see the biological development of the tumour.

"This is a really exciting prospect for researchers who are trying to understand how tumours develop."

With the unique PiggyBac system in place, it is now a case of developing multiple mice with tumours of the same type and making those crucial connections between different cancers, the mutations responsible, and, ultimately, the treatments to target those genes.

The future

At a critical moment in cancer research, the Mouse Genomics team is part of a wider effort to understand the genetic causes of cancer. New opportunities in large scale sequencing and analysis are revolutionising the information researchers can draw from human cancer samples.

"The Institute's Cancer Genome Project has ambitious plans to sequence hundreds, if not thousands, of human cancer genomes," says Roland. "With PiggyBac ready for action, we can work together with that team - using a dual approach to speed the search for and validate the genetic culprits underlying cancers."

Roland is enthusiastic about the clinical implications on the horizon in as the team enters this new era of gene discovery.

"Before I joined the Institute, my research focused on diseases of the gastrointestinal tract," explains Roland. "By developing PiggyBac, we have opened new doors to ask new questions. It is a privilege to be able to provide this tool for researchers."

As the team enters a new era of cancer gene identification and validation, Roland started by deploying PiggyBac on the hunt for genes that drive the development of intestinal and pancreatic cancers.

"Roland's dedication to developing the PiggyBac system has been remarkable," says Professor Allan Bradley, leader of the Institute's Mouse Genomics team and a pioneer in the development of mouse mutagenesis systems. "And his dedication is borne out of a determination that this research will be at the heart of clinical advances in the future."

Of course, the real rewards remain to be felt.

Targeted drugs like Herceptin, Gleevec and Iressa are proof of principle that the search for cancer genes is informing practice in the clinic. PiggyBac will now join the hunt for new cancer causing genes. And, with that precious information in hand, the pursuit will soon turn to finding drugs against those targets.

Using PiggyBac, Roland's team can develop tumour cells for teams leading drug discovery and resistance programmes. Those teams can then fire their drug compounds at the PiggyBac generated cancer cells, in the search for drugs that can stop or slow cancerous cell reproduction.

The PiggyBac project has been all-consuming for Roland and more recently Roland's wife, who joined the Institute's Mouse Genomics group soon after Roland. The pair has recently welcomed their first child to the family.

"PiggyBac has been a truly rewarding project to be involved in and has fascinated me for many years now," says Roland. "But I am also enjoying my latest foray into family life - and taking the opportunity to become familiar with the more conventional type of piggy backing!"

Suggested reading

  • PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice.

    Rad R, Rad L, Wang W, Cadinanos J, Vassiliou G, Rice S, Campos LS, Yusa K, Banerjee R, Li MA, de la Rosa J, Strong A, Lu D, Ellis P, Conte N, Yang FT, Liu P and Bradley A

    Science (New York, N.Y.) 2010;330;6007;1104-7

  • Harnessing transposons for cancer gene discovery.

    Copeland NG and Jenkins NA

    Nature reviews. Cancer 2010;10;10;696-706

  • Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice.

    Ding S, Wu X, Li G, Han M, Zhuang Y and Xu T

    Cell 2005;122;3;473-83

  • Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells.

    Ivics Z, Hackett PB, Plasterk RH and Izsvák Z

    Cell 1997;91;4;501-10

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