Seeing a New World

May I wish readers a Happy New Year – and indeed extend my felicitations to non-readers with the hope that they too will become followers! What a good idea! Not least because I suspect many are viewing the new year with a mixture of anxiety and despair. But I can promise there’s nothing like the sanity of science to restore you after a few minutes contemplating how we’re doing on the economic and political fronts.

Your starter for 2017

By happy chance a few weeks ago I tried to explain how it’s now possible to ‘re-write the manual of life’ – that is, to engineer our DNA, to fix broken genes if you like. This means that, in theory, it’s possible to correct errors in our genetic code that cause genetic diseases. As there are over 6,000 of these and they include Down syndrome, cystic fibrosis and Alzheimer’s disease, there’s no need to say it’s important. There are several ways of going about this but the one I described is called CRISPR and it’s had a lot of media coverage.

Right on cue

Well done then Keiichiro Suzuki, Juan Carlos Belmonte and friends from the Salk Institute in California together with colleagues from other centres in Spain, Saudi Arabia and China for their December paper describing a new CRISPR twist. They used a rat model of retinitis pigmentosa, a genetic disease that is a major cause of inherited blindness, afflicting about one and a half million people worldwide (one in 4,000 in the UK).

The CRISPR-Cas9 system is great but it works best in dividing cells (e.g., in skin and gut that are renewing all the time) and it’s particularly useful for knocking out genes rather than inserting new DNA. The latest modification allows a new gene to be inserted into a specific site in the DNA of cells that are not dividing (e.g., those of the eye or brain).

The bits of CRISPR-Cas9, which insert DNA at very precise locations within the genome, are delivered to target cells as part of an inert virus. However, the package also includes DNA that encourages the cells to use a repair process that can be turned on even in non-dividing cells. So CRISPR-Cas9 cuts the cell’s DNA at an exact sequence and the cell then repairs the double-strand breaks (by a process called non-homologous end joining (NHEJ) that glues the broken ends directly together). Give the cell a new bit of DNA (e.g., your favorite gene) and that will get patched in – bear in mind that the cell doesn’t ‘know’ what it’s doing: it just tries to fix damaged DNA with whatever’s at hand.

And the target?

Retinitis pigmentosa occurs when a chunk of a gene called Mertk is lost. After quite a lot of experiments to show that their method worked, Suzuki, Belmonte & Co made a viral carrier that included a normal Mertk gene and injected it under the retina of rats with the disease. After about 5 weeks the rats were making Mertk RNA as a result of the gene being correctly ‘knocked-in’ to eye cells. The light-detecting region of the eye, greatly reduced by the disease, was significantly restored, with associated appearance of MERTK protein.

      Diseased    Normal     Treated                         Diseased         Normal         Treated

pic

Left trio: Sections of the light-detecting layers of the eye in diseased (left), normal (centre) and diseased post-treatment rats (right). Right trio: corresponding fluorescence images showing MERTK expression (red: highlighted by white arrows); Cells labeled blue. (Suzuki et al. Nature 1–6 (2016) doi:10.1038/nature20565)

How did the rats see it?

Well, after treatment they were able to detect light and had significantly recovered their visual functions, albeit not to completely normal levels.

The usual caveats apply: the method isn’t hyper-efficient and a human treatment is still a long way off. Nevertheless, it’s a significant step.

The same group has also shown, using a way of re-programming the expression of just four genes, that it’s possible to arrest the signs of ageing. In other words, in mice this time, tinkering with these genes can increase lifespan – and yes, we have versions of these genes and in us they also control cell renewal.

So the New Year message is clear to see. If we can avoid turning the planet into a desert or blowing ourselves to smithereens the future is really rosy – and maybe even infinite!

References

Suzuki, K. et al. (2016). In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144-149.

Ocampo, A. et al. (2016). In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming. Cell 167, 1719–1733.

Long-live the Revolutions!!

There’s a general view that most folk don’t know much about science and, because almost day by day, science plays a more prominent role in our lives, that’s considered to be a Bad Thing. Us scientists are therefore always being told to get off our backsides and spread the word – and I try to do my bit in Betrayed by Nature, in Secret of Life (a new book shortly to be published) and in these follow-up blogs.

We may be making some progress – and, I have to admit, television has probably done more than me – though I am available (t.v. & movie head honchos please note). As one piece of evidence you could cite the way ‘DNA’ has become part of the universal lexicon, albeit often nonsensically. As evidence I call Sony Corp. Chief Executive Kazuo Hirai, as reported in The Wall Street Journal: “I’ve said this from day one. Some things at Sony are literally written into our DNA …”

Well, of course, that’s gibberish Kazuo old bean – but we know what you mean. Or do we? Most probably couldn’t tell you what the acronym stands for – but that doesn’t matter if they can explain that it’s the stuff (a ‘molecule’ would be better still!) that carries the information of inheritance and, as such, is responsible for all life. Go to the top of the class those who add that the code is in the form of chemicals called bases and there are just four of them (A, C, G & T). Something that simple doesn’t seem enough for all life but the secret is lies in the vast lengths of DNA involved. The human genome, for example, is made up of three billion letters.

A little bit of what is now history …

In the mid-1980s a number of scientists from around the world began to talk about the possibility of working out the sequence of letters that make up human DNA and thus identifying and mapping all the genes encoded by the human genome. From this emerged The Human Genome Project, a massive international collaboration, conceived in 1984 and completed in 2003. I quite often refer to this achievement as the ‘Greatest Revolution’ – meaning the biggest technical advance in the history of biology.

As that fantastic enterprise steadily advanced to its triumphant conclusion, it was accompanied by a series of mini-revolutions in technology that sky-rocketed the speed of sequencing and slashed the cost – the combined effect being an increase the efficiency of the whole process of more than 100 million-fold.

Brings us to the present …

These quite astonishing developments have continued since 2003 such that by 2009 it was possible to sequence 12 individuals in one study. By August 2016 groups from all over the world, coming together under the banner of The Exome Aggregation Consortium (ExAC), have raised the stakes 5,000-fold by sequencing no fewer than 60,706 individuals.

The name of the outfit tells you that there’s what you might think of as a very small swizz here: they didn’t sequence all the DNA, just the regions that code for proteins (exomes) – only about 1% of the three billion letters. But what highlights the power of current methods is not only the huge number of individuals sequenced but the depth of coverage – that is, the number of times each base (letter) in each individual exome was sequenced. In effect, it’s doing the same experiment so many times that errors are eliminated. Thus even genetic variants in just one person can be picked out.

seq-pic

Sequence variants between individuals. For most proteins the stretches of genomic DNA that encode their sequence  are split into regions called exons. All the expressed genes in a genome make up the exomeBy repeated sequencing The Exome Aggregation Consortium have shown that genetic variants in even one person can be reliably identified. Variants from the normal sequence found in four people are shown in red, bold letters.

It turns out that there are about 7.5 million variants and they pop up remarkably often – at one in every eight sites (bases). About half only occur once (which illustrates why DNA fingerprinting, aka DNA profiling, is so sensitive). As Jay Shendure put it, this gives us a “glimpse of the bottom of the well of genetic variation in humans.”

One of the major results of this study is that, by filtering out common variants from those associated with specific diseases, it will help to pin down the causes of Mendelian diseases (i.e. genetic disorders caused by change or alteration in a single gene, e.g., cystic fibrosis, haemophilia, sickle-cell anaemia, phenylketonuria). It’s clear that, over the next ten years, tens of millions of human genomes will be sequenced which will reveal the underlying causes of the thousands of genetic disorders.

The prize … and the puzzle

The technology is breathtaking, the amount of information being accumulated beyond comprehension. Needless to say, private enterprise has leapt on the bandwagon and you can now get your genome sequenced by, for example, 23andMe who offer “a personalised DNA service providing information and tools for individuals to learn about and explore their DNA. Find out if you are at risk for passing on an inherited condition, who you’re related to etc.” All for a mere $199!!

But you could say that the endpoint – the reason for grappling with DNA in the first place – is easy to see: eventually we will be able to define the molecular drivers of all genetic diseases and from that will follow ever improving methods of treatment and prevention.

Nevertheless, in that wonderful world I suspect we will still find ourselves brought up short by the underlying question: how one earth does DNA manage to carry the information necessary for all life?

For those who like to ponder such things, in the next piece we’ll try to help by looking at DNA from a different angle.

References

Ng, SB. et al. (2009). Nature 461, 272-276.

Lek, M. et al. (2016). Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291.

Guess Who’s Coming to Dinner?

 

Question: when is a gene not a gene? Answer: when it’s a pseudogene.

Genes are familiar enough these days when the acronym DNA has become part of everyday speech “It is in Toyota’s DNA that mistakes made once will not be repeated”, as the CEO of Toyota rather sinisterly remarked. You could say that’s pseudo-scientific rubbish but, despite that kind of liberty-taking, most will know that a gene is a stretch of our genetic material (DNA) that carries the code to make a closely related RNA molecule that, in turn, may be used as a template to make a protein ­– it’s the molecular unit of heredity. Well known too is that the Greeks gave us ‘pseudo’ – but what’s a ‘lying’ or ‘false’ gene – and who cares?

No prizes for guessing that we should all be interested because it’s emerging that pseudogenes can be important players in cancer.

Player’s biography

Pseudogenes are somewhat disreputable because they are relatives of normal genes that along the evolutionary highway have become dysfunctional by losing the capacity to be ‘expressed’ – that is, their code can no longer be transformed into RNA and protein. You could think of them as an example of the shambolic way in which species have evolved by random happenstance so that they work in their own particular niches. And if you want the outstanding example of unintelligent design, look no further than yourself, as we did in Holiday Reading (2), Poking the Blancmange.

Just for background, although it doesn’t affect the main story, there are three ways in which our genome can acquire a pseudogene:

1. A normal gene becomes functionally extinct: odd mutational events disable the stretches of DNA that control its expression. The gene is like a siding on a railway that isn’t used for years and years until eventually the points  seize up (it would be a ‘switch’ on US railroads) and the cell machinery can no longer get at it – but when this does happen we get by without that gene.

2. During evolution genes quite often get duplicated – giving multiple copies: if one of these loses its regulatory bits the duplicate gene is switched off – it’s become a ghost.

3. We owe about 8% of our genome to viruses – mainly those with RNA genomes (retroviruses) whose life-cycle turns their RNA into DNA that has then been stuck into our genome. And that’s a lot (about 100,000 bits of retrovirus DNA) especially bearing in mind that only about 1% of our genome encodes proteins.

So our precious genome is littered with corpses and fragments thereof. In the past there’s been a regrettable tendency to label this material as ‘junk’ but increasingly we’re now discovering that there may be genetic life after death, so to speak. It’s not surprising if you think about it. If random events can inactivate a gene then they might do the reverse, even if that may be a much rarer event. And indeed it’s now clear that pseudogenes can be brought back to life through the random mutational events that characterise the rough and tumble of cellular life.

So not all pseudogenes are extinct then?

Correct. Obviously we wouldn’t be wittering on about them had not some bright sparks just shown that pseudogenes – or at least one in particular – can be re-awakened to play a part in cancer. The luminaries are Florian Karreth, Pier Paolo Pandolfi and friends from all over the place (USA, UK, Italy, Singapore) who found that a pseudogene called BRAFP1 (a relative of the normal BRAF gene) can help to drive cancer development. Some earlier studies had shown that BRAFP1 was expressed (i.e. RNA was made from DNA) in various human tumours but Karreth & Co extended this, detecting significant levels of the pseudogene RNA in lymphomas and thyroid tumours and also in cells from melanoma, prostate cancer and lung cancer, whilst it’s not switched on in the corresponding normal cells.

To show that this pseudogene can drive cancers they genetically engineered its over-expression in mice, whereupon the animals developed an aggressive malignancy akin to human lymphoma (specifically diffuse large B cell lymphoma). Short-circuiting an enormous amount of work, it emerged that the pseudogene up-regulated a signaling pathway involving its normal counterpart, BRAF, that drives proliferation.

106 pic

How a pseudogene (BRAFP1) might drive cancer. Top: The scheme illustrates the ‘central dogma’ of molecular biology: DNA makes RNA makes protein. In normal cells a family of micro RNAs (different coloured wiggles) regulate the level of BRAF RNA and hence of BRAF protein (above white line).  Bottom: When the pseudogene BRAFP1 is switched on its RNA competes for the negative regulators: the result is more BRAF RNA making more BRAF protein – making cancer (Karreth et al., 2015).

Interfering RNA

The pseudogene’s RNA manages to interfere with normal control by targeting another type of RNA – micro RNAs, so called because they’re very short (about 20 bases (units) long – so they’re encoded by tiny stretches of the over 3,000 million units that make up the genome). Small they may be but there are hundreds of them and it’s become clear over the last few years that they play critical roles in regulating how much protein is made from specific RNAs. Their method is simple: they recognize (i.e. bind to) stretches of RNA that encode proteins, thereby blocking translation into protein.

Karreth & Co showed that there are about 40 different micro RNAs that can stick to the RNAs encoding BRAF or BRAFP1. Normally when there’s no (or very little) BRAFP1 around they have only BRAF to act on – and their role is to control the proliferation signal it transmits – i.e. to keep that signal to what’s required for normal cell growth control. BUT, when the pseudogene RNA is made in significant amounts the attentions of the 40 micro RNAs are divided. Result: more BRAF RNA, more BRAF protein, higher cell proliferation.

It’s a bit like you’re just sitting down to a family dinner for four when there’s a knock on the door and in walks long lost Uncle Bert, complete with wife and two kids in tow. Of course you invite them to dine too – but now a meal for four has to stretch to eight. There is something for everybody – just not as much. Similarly for the regulators of BRAF: when BRAFP1 is present there’s half as much of the RNA regulators for each – and the result, bearing mind that they are negative regulators, is that the activity of BRAF goes up and the cells proliferate more avidly. The pseudogene is driving cancer.

First but not last

For decades pseudogenes were thought of as ‘junk’ DNA along with most of the rest of the genome that didn’t encode proteins – though I might say that was a concept I never promoted. Beware labeling anything in our genome as junk for it may rise, Kraken like, to remind us of our ignorance. And, now that one pseudogene has come in from the cold and been shown to drive some cancers, you can be confident that others will follow.

References

Karreth, F.A. et al. (2015). The BRAF Pseudogene Functions as a Competitive Endogenous RNA and Induces Lymphoma In Vivo. Cell 161, 319–332.

Gosh! Wonderful GOSH

Anyone who reads these pages will long ago, I trust, have been persuaded that the molecular biology of cells is fascinating, beautiful and utterly absorbing – and all that is still true even when something goes wrong and cancers make their unwelcome appearance. Which makes cancer a brilliant topic to talk and write about – you know your audience will be captivated (well, unless you’re utterly hopeless). There’s only one snag, namely that – perhaps because of the unwelcome nature of cancers – it’s tough to make jokes. One of the best reviews I had for Betrayed by Nature was terrifically nice about it but at the end, presumably feeling that he had to balance things up, the reviewer commented that it: “..is perhaps a little too light-hearted at times…” Thank you so much anonymous critic! Crikey! If I’d been trying to do slap-stick I’d have bunged in a few of those lewd chemicals – a touch of erectone, a bit of PORN, etc. (btw, the former is used in traditional Chinese medicine to treat arthritis and the latter is poly-ornithinine, so calm down).

I guess my serious referee may have spotted that I included a poem – well, two actually, one written by the great JBS Haldane in 1964 when he discovered he had bowel cancer which begins:

I wish I had the voice of Homer

To sing of rectal carcinoma,

Which kills a lot more chaps, in fact,
Than were bumped off when Troy was sacked.

Those couplets may reflect much of JBS with whom I can’t compete but, nevertheless, in Betrayed by Nature I took a deep breath and had a go at an update that began:

Long gone are the days of Homer
But not so those of carcinoma,
Of sarcoma and leukemia

And other cancers familia.
But nowadays we meet pre-school
That great and wondrous Molecule.
We know now from the knee of Mater
That DNA’s the great creator.

and went on:

But DNA makes cancer too

Time enough—it’ll happen to you.
“No worries sport” as some would say,
These days it’s “omics” all the way.

So heed the words of JBS

Who years ago, though in distress,
Gave this advice on what to do

When something odd happens to you:
“Take blood and bumps to your physician
Whose only aim is your remission.”

I’d rather forgotten my poem until in just the last week there hit the press a story illustrating that although cancer mayn’t be particularly fertile ground for funnies it does gloriously uplifting like nothing else. It was an account of how science and medicine had come together at Great Ormond Street Hospital to save a life and it was even more thrilling because the life was that of a little girl just two years old. The saga brought my poem to mind and it seemed, though I say it myself, rather spot on.

The little girl, Layla, was three months old when she was diagnosed with acute lymphoblastic leukemia (ALL) caused by a piece of her DNA misbehaving by upping sticks and moving to a new home on another chromosome – one way in which genetic damage can lead to cancer. By her first birthday chemotherapy and a bone marrow transplant had failed and the only remaining option appeared to be palliative care. At this point the GOSH team obtained special dispensation to try a novel immunotherapy using what are being called “designer immune cells“. Over a few months Layla recovered and is now free of cancer. However, there are no reports of Waseem Qasim and his colleagues at GOSH and at University College London dancing and singing the Trafalgar Square fountains – they’re such a reserved lot these scientists and doctors.

How did they do it?

In principle they used the gene therapy approach that, helpfully, we described recently (Self Help Part 2). T cells isolated from a blood sample have novel genes inserted into their DNA and are grown in the lab before infusing into the patient. The idea is to improve the efficiency with which the T cells target a particular protein (CD19) present on the surface of the leukemia cells by giving them artificial T cell receptors (also known as chimeric T cell receptors or chimeric antigen receptors (CARs) – because they’re made by fusing several bits together to make something that sticks to the target ‘antigen’ – CD19). The engineered receptors thereby boost the immune response against the leukemia. The new genetic material is inserted into a virus that carries it into the cells. So established is this method that you can buy such modified cells from the French biotech company Cellectis.

105 picAdoptive cell transfer immunotherapy. T cells are isolated from a blood sample and novel genes inserted into their DNA. The GOSH treatment also uses gene editing by TALENs to delete two genes. The engineered T cells are expanded, selected and then infused into the patient.

Is that all?

Not quite. To give themselves a better chance the team added a couple of extra tricks. First they included in the virus a second gene, RQR8, that encodes two proteins – this helps with identifying and selecting the modified cells. The second ploy is, perhaps, the most exciting of all: they used gene editing – a rapidly developing field that permits DNA in cells to be modified directly: it really amounts to molecular cutting and pasting. Also called ‘genome editing’ or ‘genome editing with engineered nucleases’ (GEEN), this form of genetic engineering removes or inserts sections of DNA, thereby modifying the genome.

The ‘cutting’ is done by proteins (enzymes called nucleases) that snip both strands of DNA – creating double-strand breaks. So nucleases are ‘molecular scissors.’ Once a double-strand break has been made the built-in systems of cells swing into action to repair the damage (i.e. stick the DNA back together as best it can without worrying about any snipped bits – these natural processes are homologous recombination and non-homologous end-joining, though we don’t need to bother about them here).

To be of any use the nucleases need to be targeted – made to home in on a specific site (DNA sequence) – and for this the GOSH group used ‘transcription activator-like effectors’ (TALEs). The origins of these proteins could hardly be further away from cancer – they come from a family of bacteria that attacks hundreds of different types of plants from cotton to fruit and nut trees, giving rise to things like citrus canker and black rot. About six years ago Jens Boch of the Martin-Luther-University in Halle and Adam Bogdanove at Iowa State University with their colleagues showed that these bugs did their dirty deeds by binding to regulatory regions of DNA thereby changing the expression of genes, hence affecting cell behavior. It turned out that their specificity came from a remarkably simple code formed by the amino acids of TALE proteins. From that it’s a relatively simple step to make artificial TALE proteins to target precise stretches of DNA and to couple them to a nuclease to do the cutting. The whole thing makes a TALEN (transcription activator-like effector nuclease). TALE proteins work in pairs (i.e. they bind as dimers on a target DNA site) so an artificial TALEN is like using both your hands to grip a piece of wood either side of the point where, using your third hand, you make the cut. The DNA that encodes the whole thing is inserted into plasmids that are transfected into the target cells; the expressed gene products then enter the nucleus to work on the host cell’s genome. There are currently three other approaches to nuclease engineering (zinc finger nucleases, the CRISPR/Cas system and meganucleases) but we can leave them for another time.

The TALENs made by the GOSH group knocked out the T cell receptor (to eliminate the risk of an immune reaction against the engineered T cells (called graft-versus-host disease) and CD52 (encodes a protein on the surface of mature lymphocytes that is the target of the monoclonal antibody alemtuzumab – so this drug can be used to prevent rejection by the host without affecting the engineered T cells).

What next?

This wonderful result is not a permanent cure for Layla but it appears to be working to stave off the disease whilst she awaits a matched T cell donor. It’s worth noting that a rather similar approach has been used with some success in treating HIV patients but it should be born in mind that, brilliant though these advances are, they are not without risks – for example, it’s possible that the vector (virus) that delivers DNA might have long-term effects – only time will tell.

Almost the most important thing in this story is what the GOSH group didn’t do. They used the TALENs gene editing method to knock out genes but it’s also a way of inserting new DNA. All you need to do is add double-stranded DNA fragments in the correct form at the same time and the cell’s repair system will incorporate them into the genome. That offers the possibility of being able to repair DNA damage that has caused loss of gene function – a major factor in almost all cancers. Although there is still no way of tackling the associated problem of how to target gene editing to tumour cells, it may be that Layla’s triumph is a really significant step for cancer therapy.

Reference

Smith, J. et al. (2015). UCART19, an allogeneic “off-the-shelf” adoptive T-cell immunotherapy against CD19+ B-cell leukemias. Journal of Clinical Oncology 33, 2015 (suppl; abstr 3069).

 

Lethal ZIP codes

In Keeping Cancer Catatonic we retailed how, over 125 years ago, the London physician Stephen Paget came up with his ‘seed and soil’ idea to explain why it was that when cancers spread to distant sites around the body by getting into the circulation they didn’t simply stick to the first tissue they came across. Paget had spotted that cancers tend to have preferred sites for spreading: tumours of the eye tend to travel to the liver, rather than the much handier brain, and breast cancers, Paget’s speciality, commonly spread to the liver but also to the lungs, kidneys, spleen and bone. So his idea was that certain distant secondary sites are somehow made more receptive to tumor growth, just as soil can be prepared for seeds to sprout.

So the key question became ‘how?’ and it’s hung in the cancer air for well over a century during which we’ve made very little progress towards an answer – and it is crucial because the business of tumour cells spreading (metastasizing) causes most cancer deaths (over 90%).

But, at long last, things have started to move, largely due to the efforts of David Lyden and his colleagues at Weill Cornell Medical College. Their first astonishing contribution was to show that cells in primary tumours release messengers into the circulation and these, in effect, tag what will become landing points for wandering tumour cells – i.e., the target sites are determined before any tumour cells actually set foot outside the confines of the primary tumour.

After that seismic revelation the story advanced a step further (in Scattering the Bad Seed) with some molecular detail of how the sites are marked – an effect Lyden has christened ‘Bookmarking cancer’ – and how when tumour cells do settle in their new niche they may be kept dormant for many years before starting to expand.

Carrying the flag

The next chapter in the story, as retailed in Holiday Reading (4) – Can We Make Resistance Futile?, revealed that the message is carried by small sacs – like little cells – called exosomes that are released from tumour cells. These float around the circulation until they find their target site, whereupon they plant the flag by setting off a chain reaction that produces a sticky protein – fibronectin – a kind of glue for immune cells and tumour cells.

That is all truly amazing stuff but, as we noted in Holiday Reading (4) – Can We Make Resistance Futile?, a recurring theme in science is that one answer merely poses the next question – in this case ‘what’s the messenger?’

As in all the best thrillers, the authors have kept us in suspense to the last, helped presumably by their not knowing the answer. But in this week’s Nature (Oct. 28, 2015) comes the denoument to this whodunit.

Mister postman look and see …

Many moons ago an outfit called the Marvelettes had a No. 1 hit with Please Mr. Postman and somewhat later the Fab Four did a re-hash that met with equal success. Perhaps we should have asked them how nature would go about directing little packages around the body. John, Ringo and the lads would, with their earthy, Liverpudlian logic, have pointed out the triviality of the problem of exosome addressing. ‘It’s not like you’re sending stuff all over the world, is it? You’ve only got a few targets – the major organs of the body. So a dead simple code will do. You know your messengers are proteins – ’coz they do everything – OK? So, pick a protein that comes in two bits with a few variants of each: mix and match and there’s yer postcodes. Now … what was that ditty about yellow subsurface vessels …’

And so it came to pass …

And the messenger is …

A family of proteins called integrins whose job is to span the membranes of cells, thereby promoting cell-cell interactions. They are indeed made of two different chains stuck together (called α (alpha) and β (beta)) and the upshot is that our cells can make about 24 unique integrins – more than enough to form a coded address system to direct tumour cells around the body. Well done lads!

What Ayuko Hoshino, David Lyden and their many collaborators did was to tag exosomes released from various types of cancer cell with a fluorescent dye and inject them into mice. The fluorescent label enabled them to track the exosomes and it turned out that, for a variety of cancer cells (breast, pancreatic, colorectal, lung, melanoma and pediatric) the exosomes travelled to the organs associated with metastasis (e.g., breast cancer exosomes stuck in the lungs, pancreatic cancer exosomes in the liver, etc). In other words exosome spread mimicked the pattern of the tumour from which they were derived. Once they had landed the exosomes set about reprogramming the organ sites to make a fertile microenvironment capable of supporting tumor cell growth in a new colony.

When they looked at the exosome proteins they found a particular member of the integrin family flagged each organ-specific site. Thus α6β4 promotes lung metastasis, αvβ5 homes in on the liver, αvβ3 on the brain, etc.

MapFinding a home

To spread around the body (metastasise) primary tumours first release small sacs (exosomes) carrying protein tags (integrins). Moving through the circulatory system the integrin tags home in to specific addresses found on different organs. The effect of exosomes sticking to target sites is to prepare the ground for cells released by the tumour to adhere and colonise.

Down the tube

You could think of primary tumours as being a bit like us when we move to a new city and try to find a des. res. in a place you don’t know. We could just ramble round the subway system until something catches our eye but that might take for ever. Much more efficient is to ask someone with local knowledge where would be good spots to target. For disseminating tumours their exosomes are the scouts who do the foot-slogging: the protein signatures on the surface of these small, tumour-secreted packages home in on postcodes that define a desirable locale for metastatic spread.

Shooting the messenger

An obvious question is ‘If exosomes are critical in defining metastatic sites, can you block their action – and what happens when you do?’ In preliminary experiments Hoshino & Co showed that either knockdown of specific integrins or blocking the capacity of these proteins to stick to their targets (with a specific antibody or short synthetic peptides) significantly reduced exosome adhesion, thereby blocking pre-metastatic niche formation and liver metastasis.

A new beginning?

We described these fabulous results as the denouement but, of course, it isn’t. As Mr. Churchill remarked in a somewhat different context: ‘Now this is not the end.’ It is rather a step to answering an old question but it’s incredibly exciting. If screening for exosomes leads to the detection of cancer not just years but perhaps decades earlier than can be achieved by present methods and if blocking their action can keep metastasis at bay, then the field of cancer will be utterly transformed.

References

Hoshino, A. et al. (2015). Tumour exosome integrins determine organotropic metastasis. Nature doi:10.1038/nature15756.

Ruoslahti, E. (1996). RGD and Other Recognition Sequences for Integrins. Annual Review of Cell and Developmental Biology 12, 697-715.

Mutating into Gold

It’s probably just as well that few us are aware that the bodies we live in are a battlefield – the cells and molecules that make us are in constant strife to ensure our survival. The lid is lifted from time to time – when we get a cold or pick up some other infection and our immune response sorts it out but not without giving us a headache or a runny nose, just to let us know it’s on the job. By and large though, we plough our furrow in glorious ignorance.

Saving our cells

Perhaps the most important of all the running battles is to save our DNA – that is, to repair the damage continuously suffered by our genetic material so we can carry on. It’s an uphill struggle. The DNA in one of our cells can take up to a million hits every day – and the bombardment comes from every direction: from radiation, air pollution and carcinogens in some of the food we eat. And, of course, we don’t need to mention cigarette smoke.

Damaged chromosomes (blue arrows)

Damaged chromosomes    (blue arrows)

On top of all that cells have to make a new DNA copy every time they reproduce – and we do a lot of that: recall that you set sail on the journey of life as one single, fertilized egg cell and now look at you: a clump of ten trillion (1013) cells that, just to stay as you are, has to make one million new cells every second. What’s more some of your cells deliberately break their own DNA in a process called ‘gene shuffling’ that goes to make the finished product of your aforementioned immune system. The biochemical machinery that does these jobs is mighty efficient but nobody’s perfect – except, of course, for John Eales, Australia’s most successful rugby union captain, nicknamed “Nobody” because “Nobody’s perfect”. When the three thousand million base-pairs of DNA are stuck together for a new cell there’s a mistake about once in every million units added – but a kind of quality control check (mismatch repair) then fixes most of these, so that the overall error is about one in a thousand million. That’s one example of the nifty ways evolution has come up with to fix the damage suffered by our genetic material from all this replicating, assaulting and constructing.

Keeping the show on the road

The overall upshot of the repair machinery is that less than one mutation per day becomes fixed in our genomes – and thus passed on to succeeding generations of cells. The range of things that can damage DNA – and hence the different forms that damage can take – tells you that there must be several different repair systems and indeed we now know that about 200 genes and their protein products have a hand in some repair process or another. There’s so much to know that DNA damage and repair has its own data-base called, inevitably, REPAIRtoire. Much of what we know is, to a considerable extent, thanks to the labours of Tomas Lindahl, Paul Modrich and Aziz Sancar who have just been jointly awarded this year’s Nobel Prize in Chemistry. Because damage to DNA – aka mutations – drives the development of cancers you might suppose that in these pages we will have met these gentlemen before – and indeed we have, if not by name.

Tomas Lindahl Paul Modrich Aziz Sancar

Tomas Lindahl                      Paul Modrich                       Aziz Sancar

Winners of the 2015 Nobel Prize in Chemistry

Forty odd years ago much of the above would have bewildered cell biologists. Thirty years before then, in 1944, Oswald Avery, Colin MacLeod and Maclyn McCarty had shown for the first time that genes are composed of DNA, a finding confirmed in 1952 by Alfred Hershey and Martha Chase in a classic experiment using a virus that infects and replicates within a bacterium. But with the acceptance that, however improbable, our genetic material was indeed made of DNA there came the assumption that it must be very stable. After all, if it carried our most valuable possession then surely it had to be made of molecular granite, absolutely resistant to any kind of chemical change or degradation. Had the bewildered boffins been told that in the twenty-first century we would be sequencing woolly mammoth DNA from samples that are millions of years old they would have been confirmed in their view.

It was Tomas Lindahl in the early 1970s who demonstrated that, although DNA is indeed more stable than its close rello RNA (the intermediate in making proteins) it nevertheless decays quite rapidly under normal conditions – it’s only when sealed in permafrost or blobs of amber that it becomes frozen in time. Lindahl realized that for life based on DNA to have evolved there had to be repair systems that could sustain our genetic material in a functional state and he went on to resolve how one of these did it. Aziz Sancar has worked particularly on the circadian clock (discovering that CRY is a clock protein) and how cells repair ultraviolet radiation damage to DNA: people born with defects in this system develop skin cancer if they are exposed to sunlight. Paul Modrich has contributed mainly to our knowledge of mismatch repair.

Lindahl, Modrich, Sancar and their colleagues over many years haven’t come up with the philosopher’s stone – the chemists still can’t transmute base metals into gold without the aid of a particle accelerator. But what they have done is much more useful for mankind. Revealing the detail of how genome maintenance works has already lead to new cancer treatments and from this beginning will come greater benefits as time goes by. They should enjoy the proceeds of turning molecular knowledge if not to gold then into Swedish kronor (8 million of them) – for the rest of the world it’s a bargain.

References

Lindahl, T. (1993). Instability and decay of the primary structure of DNA. Nature 362, 709-715.

Yang YG, Lindahl T, Barnes DE. (2007). Trex1 exonuclease degrades ssDNA to prevent chronic checkpoint activation and autoimmune disease. Cell 131, 873-886.

Shao, H, Baitinger, C, Soderblom, EJ, Burdett, V, and Modrich, P. (2014). Hydrolytic function of Exo1 in mammalian mismatch repair. Nucleic Acids Research 42, 7104-7112.

Tan C, Liu Z, Li J, Guo X, Wang L, Sancar A, Zhong D. (2015). The molecular origin of high DNA-repair efficiency by photolyase. Nat Commun. 6, 7302.

Taking a Swiss Army Knife to Cancer

Murder is easy. You just need a weapon and a victim. And, I guess the police would add, opportunity. I hasten to point out that’s an observational note rather than an autobiographical aside. It’s relevant here because treating cancer is intentional homicide on a grand scale – the slaughter of millions of tumour cells. For individuals we cannot say whether the perfect murder is possible – how would we know – but on the mass scale history shows that even the most efficient machines for genocide have been, fortunately, less than perfect. In other words through incredible adaptability, ingenuity, determination and sheer will power, some folk will survive even the most extreme efforts of their fellow men to exterminate them. Cancers tend to mirror their carriers. With only rare exceptions, whatever we throw at them in attempts to eliminate their unwelcome presence, some of the little blighters will dodge the bullets, take a deep breath and start reproducing again. ‘What doesn’t kill you makes you stronger’ as the American chanteuse Kelly Clarkson has it – though to be fair I think she pinched the line from Nietzsche.

Multiple whammys

For cancer cells dodging extinction requires adaptability: using the flexibility of the human genetic code enshrined in DNA to change the pattern of gene expression or to develop new mutations that short-circuit the effects of drugs – finding many different ways to render useless drugs that worked initially. You might draw a parallel with the idea, correct as it turned out, of the prisoners who staged The Great Escape from Stalag Luft III in World War II that if they initially dug three tunnels simultaneously (Tom, Dick and Harry) the guards might find one but they’d probably not find all three.

An approach increasingly permeating cancer therapy is how to target several escape routes at once – can we at least give the tumour cell a serious headache in the hope that while it’s grappling with a molecular carpet bombing it might be more likely to drop dead. One way of doing this is simply to administer drug combinations and this has met with some success. However, for the most part, agents are not specific for tumour cells and their actions on normal cells give rise to the major problem of side effects.

Step forward Yongjun Liu and colleagues from Shandong University, Jinnan, People’s Republic of China and the sophisticated world of chemistry with efforts to fire a broad-shot that combines different ways of killing tumour cells with at least some degree of specific targeting.

Making the bullets

These chemists are clever chaps but, taken one step at a time, what they’ve done to make a very promising agent is simple. The game is molecular Lego – making a series of separate bits then hooking them together. The trendy name is click chemistry, a term coined in 1998 by Barry Sharpless and colleagues at The Scripps Research Institute, to describe reactions in which large, pre-formed molecules are linked to make even more complex multi-functional structures. You could describe proteins as a product of ‘click chemistry’ as cells join amino acid units to make huge chains – but you wouldn’t as it’s better to keep the name for synthetic reactions that make novel modules.

It might help to recall some school chemistry:

acid + base = salt + water (e.g., HCl + NaOH = NaCl + H2O)

Click chemistry is the same idea but the reactants are large molecules, rather than atoms of hydrogen, chlorine and sodium.

Anti-freeze to anti-cancer in a couple of clicks

The starting point here is remarkably familiar – it’s antifreeze, a chemical added to cooling systems to lower the freezing point of water (e.g., in motor engines). Antifreeze is ethylene glycol (two linked atoms of carbon with hydrogens: HO-CH2-CH2-OH): make a string of these molecules and you have a polymer – poly-ethylene glycol (PEG).

For click chemists it’s easy to tag things on to biologicial molecules, including PEG and most proteins. This study used biotin – a vitamin that works like a molecular glue by sticking strongly to another small molecule called avidin, found in egg white. Avidin can therefore be used to fish for anything tagged with biotin – it simply hooks two biotins together. The protein used here is an antibody that binds to a signaling molecule (VEGFR) present on the surface of most tumour cells and blood vessels. VEGFR helps tumour growth by providing a new blood supply – an effect blocked when the antibody binds to it.

Sounds familiar?

If chains of carbon atoms decorated with hydrogens seem familiar, so they should. They’re fats (the saturated fats you get in cream and butter are very similar to the chains of PEG). As anyone who’s done the washing up knows, fats and water don’t get on (which is why we have detergents). Put them in water and fats huddle together in blobs called micelles – sacs of fat. This gives them a useful property: if you mix something else in the water – a drug for instance – and then add PEG and separate the micelles that form, you’ve got drug trapped in a kind of carrier bag. Often called nanoparticles, these small, molecular bubbles made by chemists are packets of drug ready to be delivered.

Micelle Blog picA sac of poly-ethylene glycol (PEG) with entrapped drug (red dots) tagged in three different ways (Liu et al., 2014).

Addressing the parcel

To turn PEG into a parcel two chemical tricks are needed. The first is to tag PEG with biotin. Now the nanoparticles will pick up VEGFR antibody labeled with avidin – and the antibody label can target the micelles to tumour cells and blood vessels.

Exploding the package

The second trick is the addition of another polymer (a chain of histidine amino acids) that triggers the disassembly of the nanoparticles when they find themselves close to or inside tumour cells – a more acidic environment than the circulation.

Seeing the results

The final twist is to include another modified PEG – this with a chemical group that binds gadolinium when it’s added to the water. Gadolinium is an ion (Gd3+) which shows up brightly in MRI scans – the idea being to highlight where the nanoparticles end up after injection into animals.

Does it work?

These multicomponent nanoparticles resemble a Swiss Army knife – all sorts of gadgets sticking out all over the place: PEG to make sacs that contain a drug, biotin hooked to VEGFR antibody to home in on tumour cells, an acidity sensor so the thing falls apart and releases its content on arrival and a contrast enhancer that shows up where this is happening in an MRI scan.

Injected into mice with liver tumours, these multi-functional nanoparticles do indeed home in on the tumours and their surroundings and drastically reduce tumour growth when they carry the drug sorafenib. Sorafenib is the only agent that has been shown to affect liver cancers, although its effects are brief. Compared to sorafenib alone, these new nanoparticles are about three times more potent – presumably because of their targeted delivery.

Where are we?

This wonderfully clever chemistry will not cure liver cancer. A good result when it reaches human trials would be six months remission by comparison with the current average of two months from treatment with sorafenib alone. But what it does show is that hitting cancers hard in multiple ways at least slows them down. We can only hope that more potent drugs and further ingenuity will progressively extend this capacity. The end is not in sight but brilliant technical advances such as that from Yongjun Liu’s lab may be spotlighting the way ahead.

Reference

Yongjun Liu et al., (2014). Multifunctional pH-sensitive polymeric nanoparticles for theranostics evaluated experimentally in cancer. Nanoscale 6, 3231-3242.

The Hay Festival

According to the Hay Festival  a recording of my talk ‘Demystifying Cancer’ on Wednesday 28th May should be available on their web site shortly and it can also be heard on the university site. However, I thought it might be helpful to post a version, not least for the for the rather breathless lady who arrived at the book signing session apologising for missing the lecture because she’d got stuck in mud. So for her and perhaps for many others I had the privilege of chatting to afterwards, read on …

 The Amazing World of Cells, Molecules … and CancerOpening pic

One of the biggest influences on my early years was the composer and conductor Antony Hopkins, who died a few days ago. Most of what I knew about music by the time I was 15 came from his wonderfully clear dissections of compositions in the series Talking About Music broadcast by the BBC Third Programme. When he was axed by the Beeb in 1992 for being ‘too elitist’ – yes, they talked that sort of drivel even then – Hopkins might have wished he’d been a biologist. After all, biology must be the easiest subject in the world to talk about. Your audience is hooked from the outset because they know it’s about them – if not directly then because all living things on the planet are interlinked – so even the BBC would struggle to make an ‘elitism’ charge stick. They know too that it’s beautiful, astonishing and often funny – both from what they see around them and also, of course, courtesy of David Attenborough. So it’s not a surprise when you show them that the micro-world of cells and molecules is every bit as wonderful.

The secret of life

What does come as a bit of a shock to most non-scientists is when you explain the secret of life. No, that’s not handing round pots of an immortalization elixir – much better, it’s outlining what’s sometimes rather ponderously called the central dogma of molecular biology – the fact that our genetic material (aka DNA) is made from only four basic units (most easily remembered by their initials: A, C, G and T – humans have over three thousand million of these stuck together). This is our ‘genome’ and the ‘genetic code’ enshrined in the DNA sequence makes us what we are – with small variations giving rise to the differences between individuals. The genetic code carries instructions for glueing together another set of small chemicals to make proteins. There are 20 of these (amino acids) and they can be assembled in any order to make proteins that can be thousands or even tens of thousands of amino acids long. These assemblies fold up into 3D shapes that give them specific activities. Proteins make living things what they are – they’re ‘the machines of life’ – and their infinite variety is responsible for all the different species to have appeared on earth. Can the basis of life really be so simple?

The paradox of cancer

Turning to cancer, a three word definition of ‘cells behaving badly’ would do fine. A more scientific version would be ‘cells proliferating abnormally.’ That is, cells reproducing either when they shouldn’t, or more rapidly than normal, or doing so in the wrong place. The cause of this unfriendly behavior is damaged DNA, that is, alteration in the genetic code – any such change being a ‘mutation’. If a mutation affects a protein so that it becomes, say, hyperactive at making cells proliferate (i.e. dividing to make more cells), you have a potential cancer ‘driver’. So at heart cancer’s very simple: it’s driven by mutations in DNA that affect proteins controlling proliferation. That’s true even of the 20% or so of cancers caused by chronic infection – because that provokes inflammation, which in turn leads to DNA damage.

The complexity of cancer arises because, in contrast to several thousand other genetic diseases in which just a single gene is abnormal (e.g., cystic fibrosis), tumour cells accumulate lots of mutations. Within this genetic mayhem, relatively small groups of potent mutations (half a dozen or so) emerge that do the ‘driving’. Though only a few ‘driver mutations’ are required, an almost limitless number of combinations can arise.

Accumulating mutations takes time, which is why cancers are predominantly diseases of old age. Even so, we should be aware that life is a game of genetic roulette in which each individual has to deal with the dice thrown by their parents. The genetic cards we’re dealt at birth may combine with mutations that we pick up all the time (due to radiation from the sun and the ground, from some foods and as a result of chemical reactions going on inside us) to cause cancers and, albeit rarely, in unlucky individuals these can arise at an early age. However, aside from what Mother Nature endows, humans are prone to giving things a helping hand through self-destructive life-style choices – the major culprits, of course, being tobacco, alcohol and poor diets, the latter being linked to becoming overweight and obese. Despite these appalling habits we’re living longer (twice as long as at the beginning of the twentieth century) which means that cancer incidence will inevitably rise as we have more time to pick up the necessary mutations. Nevertheless, if we could ban cigarettes, drastically reduce alcohol consumption and eat sensibly we could reduce the incidence of cancers by well over a half.

How are we doing?

Some readers may recall that forty-odd years ago in 1971 President Nixon famously committed the intellectual and technological might of the USA to a ‘War on Cancer’ saying, in effect, let’s give the boffins pots of money to sort it out pronto. Amazing discoveries and improved treatments have emerged in the wake of that dramatic challenge (not all from Uncle Sam, by the way!) but, had we used the first grant money to make a time machine from which we were able to report back that in 2013 nearly six hundred thousand Americans died from cancer, that the global death toll was over eight million people a year and will rise to more than 13 million by 2030 (according to the Union for International Cancer Control), rather less cash might subsequently have been doled out. Don’t get me wrong: Tricky Dicky was spot on to do what he did and scientists are wonderful – clever, dedicated, incredibly hard-working, totally uninterested in personal gain and almost always handsome and charming. But the point here is that, well, sometimes scientific questions are a little bit more difficult than they look.

Notwithstanding, there have been fantastic advances. The five year survival rates for breast and prostate cancers have gone from below 50% to around 90% – improvements to which many factors have contributed including greater public awareness (increasing the take-up of screening services), improved surgical and radiology methods and, of course, new drugs. But for all the inspiration, perspiration and fiscal lubrication, cancer still kills over one third of all people in what we like to refer to as the “developed” world, globally breast cancer killed over half a million in 2012 and for many types of cancer almost no impact has been made on the survival figures. In the light of that rather gloomy summary we might ask whether there is any light at the end of the tunnel.

The Greatest Revolution

From one perspective it’s surprising we’ve made much progress at all because until just a few years ago we had little idea about the molecular events that drive cancers and most of the advances in drug treatment have come about empirically, as the scientists say – in plain language by trial and error. But in 2003 there occurred one of the great moments in science – arguably the most influential event in the entire history of medical science – the unveiling of the first complete DNA sequence of a human genome. This was the product of a miraculous feat of international collaboration called The Human Genome Project that determined the order of the four units (A, C, G and T) that make up human DNA (i.e. the sequence). Set up in 1990, the project was completed by 2003, two years ahead of schedule and under budget.

If the human genome project was one of the most sensational triumphs in the history of science what has happened in the ensuing 10 years is perhaps even more dazzling. Quite breathtaking technical advances now mean that DNA can be sequenced on a truly industrial scale and it is possible to obtain the complete sequence of a human genome in a day or so at a cost of about $1,000.

These developments represent the greatest revolution because they are already having an impact on every facet of biological science: food production, microbiology and pesticides, biofuels – and medicine. But no field has been more dramatically affected by this technological broadside than cancer and already thousands of genomes have been sequenced from a wide range of tumours. The most striking result has been to reveal the full detail of the astonishing genetic mayhem that characterizes cancer cells. Tens of thousands or even hundreds of thousands of mutations featuring every kind of molecular gymnastics imaginable occur in a typical tumour cell, creating a landscape of stunning complexity. At first sight this makes the therapeutic challenge seem daunting, but all may not be lost because the vast majority of this genetic damage plays no role in cancer development (they’re ‘passenger’ mutations) and the power of sequencing now means they can be sifted from the much smaller hand of ‘driver’ mutations. From this distillation have emerged sets of ‘mutational signatures’ for most of the major types of cancers. This is a seismic shift from the traditional method of assessing tumours – looking directly at the cells after treating them with markers to highlight particular features – and this genetic approach, providing for the first time a rigorous molecular basis for classifying tumours, is already affecting clinical practice through its prognostic potential and informing decisions about treatment.

A new era

One of the first applications of genomics to cancer, was undertaken by a group at The Wellcome Trust Sanger Institute near Cambridge (where the UK part of the Human Genome Project had been carried out), who screened samples of the skin cancer known as malignant melanoma. This is now the fifth most common UK cancer – in young people (aged 15 to 34) it’s the second most common – and it killed over 2,200 in 2012. Remarkably, about half the tumours were found to have a hyperactivating mutation in a gene called BRAF, the effect being to switch on a signal pathway so that it drives cell proliferation continuously. It was a remarkable finding because up until then virtually nothing was known about the molecular biology of this cancer. Even more amazingly, within a few years it had lead to the development of drugs that caused substantial regression of melanomas that had spread to secondary sites (metastasized).

This was an early example of what has become known as personalized medicine – the concept that molecular analysis will permit treatment regimens to be tailored to the stage of development of an individual’s cancer. And maybe, at some distant time, the era of personalized medicine will truly come about. At the moment, however, we have very few drugs that are specific for cancer cells – and even when drugs work initially, patients almost invariably relapse as tumours become resistant and the cancer returns – one of the major challenges for cancer biology.

It behoves us therefore to think laterally, of impersonal medicine if you like, and one alternative approach to trying to hit the almost limitless range of targets revealed by genomics is to ask: do tumour cells have a molecular jugular – a master regulator through which all the signals telling it to proliferate have to pass. There’s an obvious candidate – a protein called MYC that is essential for cells to proliferate. The problem with stopping MYC working is that humans make about one million new cells a second, just to maintain the status quo – so informed opinion says that blocking MYC will kill so many cells the animal will die – which would certainly fix cancer but not quite in the way we’re aiming for. Astoundingly, it turns out in mice at least it doesn’t work like that. Normal cells tolerate attenuation of MYC activity pretty well but the tumour cells die. What a result!! We should, of course, bear in mind that the highway of cancer therapy is littered with successful mouse treatments that simply didn’t work in us – but maybe this time we’ll get lucky.

An Achilles’ heel?

In defining cancers we noted the possibility that tumour cells might proliferate in the wrong place. So important is this capacity that most cancer patients die as a result of tumour cells spreading around the body and founding secondary colonies at new sites – a phenomenon called metastasis. Well over 100 years ago a clever London physician by the name of Stephen Paget drew a parallel between the growth of tumours and plants: ‘When a plant goes to seed, its seeds are carried in all directions; but they can only live and grow if they fall on congenial soil.’ From this emerged the “seed and soil” theory as at least a step to explaining metastasis. Thus have things languished until very recent findings have begun to lift the metastatic veil. Quite unexpectedly, in mouse models, primary tumours dispatch chemical messengers into the blood stream long before any of their cells set sail. These protein news-bearers essentially tag a landing site within the circulatory system on which the tumour cells touch down. Which sites are tagged depends on the type of tumour – consistent with the fact that human cancers show different preferences in metastatic targets.

These revelations have been matched by stunning new video methods that permit tumour cells to be tracked inside live mice. For the first time this has shone a light on the mystery of how tumour cells get into the circulation – the first step in metastasis. Astonishingly tumour cells attach themselves to a type of normal cell, macrophages, whose usual job is to engulf and digest cellular debris and bugs. The upshot of this embrace is that the macrophages cause the cells that line blood vessels to lose contact with each other, creating gaps in the vessel wall through which tumour cells squeeze to make their escape. This extraordinary hijacking has prognostic value and is being used to develop a test for the risk of metastasis in breast cancers.

The very fact that cancers manifest their most devastating effects by spreading to other sites may lay bare an Achilles’ heel. Other remarkable technical developments mean that it’s now possible to fish out cancer cells (or DNA they’ve released) from a teaspoonful of circulating blood (that’s a pretty neat trick in itself, given we’re talking about fewer than 100 tumour cells in a sea of several billion cells for every cubic millimeter of blood). Coupling this to genome sequencing has already permitted the response of patients to drug therapy to be monitored but an even more exciting prospect is that through these methods we may be moving towards cancer detection perhaps years earlier than is possible by current techniques.

As we’ve seen, practically every aspect of cancer biology is now dominated by genomics. Last picIt’s so trendy that anyone can join in. Songs have been written about DNA and you can even make a musical of your own genetic code, French physicist Joel Sternheimer having come up with a new genre – protein music – in which sequence information is converted to musical notes. Antony Hopkins, ever receptive to new ideas, would have been enthralled and, with characteristic enthusiasm, been only too happy to devote an episode of Talking About Music to making tunes from nature.

A Refresher from the BBC

Regular readers will probably feel they know all this stuff but if you’re interested in a spirited and wide-ranging conversation about cancer with the wonderful Jeremy Vine on his BBC Radio 2 show yesterday you can find it at:

http://www.bbc.co.uk/programmes/b03yn0jd about 1 hour 10 min from the beginning.

BBC Radio 4As ever, any arising thoughts, questions or comments appreciated – apart, of course, from the below the belt: “Judging by the photo it’s a good job it was radio not t.v.”

 

Seeing the Invisible: A Cancer Early Warning System?

Sherlock Holmes enthusiasts who also follow this column may, in a contemplative moment, have asked themselves whether their hero would have made a good cancer detective. Answer perhaps ‘yes’ in that he was obsessive about sticking to the facts and not guessing and would probably have said that, when tracking down a secretive quarry, you need to be as open-minded as possible in looking for clues. One of his most celebrated efforts at marrying observation with knowledge was his greeting upon first meeting Dr. Watson: “How are you? You have been in Afghanistan, I perceive”. Watson was suitably astonished by this apparent clairvoyance although its basis was in fact rather mundane and only beyond him because, as Sherlock kindly explained, “You see, but you do not observe.”

Holmes-Image-Loupe

Dr. Holmes perchance?

If Watson had paused to wonder whether Holmes’ combination of superiority complex and investigative genius would have fitted him for a career in the medical fraternity, he might have reflected that indeed many internal afflictions do manifest external signs – much as the furtive body language of a felon on a job might mark him out to the observant eye in the throng of bodies pressing into Baker Street underground station. So perhaps the ’tec turned doc could make it in infectious diseases or become a consultant in rheumatoid arthritis. But would he have steered clear of oncology, reasoning that most cancers are without symptoms during their early development and that even he could not observe the invisible?

Lithograph of Baker Street Station   Baker Street Station on the Metropolitan Railway in 1863 (London Transport Museum collection)

Probably, but before taking that decision he would have asked for a tutorial – perhaps from that bright fellow Stephen Paget, who would have explained that cancers are unusual lumps of cells that can often be cut out by surgeons such as himself. But he’d have highlighted the problem that similar growths commonly turn up later at other, secondary, sites in the body – they are what kills most cancer patients and no one has a clue how this happens or what to do about it. Holmes would doubtless have taken a deep suck on his pipe, commented that, as no one appeared to disagree with William Harvey’s 250 year old finding that blood is passed to every nook and cranny of the body by the circulatory system, it scarcely required his giant intellect to deduce that to be the most probable way of spreading tumours. Further observing that cancers develop very slowly, he would have pointed out that it is highly likely that within the body there might be clues – molecular signs that something is amiss – long before overt disease appears. All that was required was a biological magnifying glass and tweezers to spot and pick out rogue cells and molecules. Muttering ‘Elementary’ he would then have asked to be excused to return to the really tricky problem of outsmarting Professor Moriarty.

An Achilles’ heel?

Well, as we have just reviewed in Scattering the Bad Seed, some 130 years after that imaginary encounter the ‘elementary’ way in which tumours spread to form metastases is just beginning to be revealed and, of course, the hope is that eventually this knowledge will lead to ways of treating disseminated cancers or even preventing them. That’s a wonderful prospect but even more exciting are technical advances enabling us to exploit what Sherlock had spotted as something of a cancer Achilles’ heel – namely that, if tumour cells spread via the bloodstream, we need only the right tools (magnifying glass and tweezers) to detect secondary growths almost before they’ve started to form. As most people know, the earlier cancers are caught the more likely they are to be cured, the most critical intervention being before they have spread to form metastases that are the major cause of death.

The things you find in blood

In fact, quite apart from intact tumour cells migrating around the circulation, it’s been known for 40 years that most types of cell in our bodies have the rather odd quirk of releasing short bits of their DNA into the circulation. Cancer cells do this too and these chromosome fragments reflect the genetic mayhem that is their hallmark. How DNA gets out of the nucleus and then across the outer membrane of the cell isn’t known but it does – and the bits of nucleic acid act as messengers, being taken up by other cells that respond by changing their behaviour. In Beware of Greeks we saw that DNA fragments released by leukemia cells can help those cells escape from the bone marrow into circulating blood.

There’s yet another sort of cellular garbage swishing around in our circulation: small sacs like little cells that contain proteins and RNAs (nucleic acids closely related to DNA). These small, secreted vesicles are called exosomes and in fact they’re not at all rubbish but are also messengers, communicating with other cells by fusing and transferring their contents. So exosomes are another form of environmental educator.

Going fishing

The problem has been that until very recently it has not been possible to fish out tumour cells or DNA from the vast number of cells in blood (we’ve each got over 20 trillion red blood cells in our five litres or so). However, an exciting new development has been the application of silicon chip technology to the detection of circulating tumour cells (CTCs). The chips, which are the size of a microscope slide (10 x 2 cm), have about 80,000 microscopic columns etched on their surface that are coated with an array of antibodies that stick to molecules expressed on the surface of CTCs. By incorporating the chips into small flow cells it’s possible to capture about 100 CTCs from a teaspoon of blood – that’s pulling out one tumour cell from a background of a billion (109) normal cells.

CTC CHIP

Tumour cell isolation from whole blood by a CTC-chip. Whole blood is circulated through a flow cell containing the capture columns (Stott et al., 2010)

This microfluidics approach can also be used to isolate tumour cell DNA. For this the coatings are short stretches of artificial DNA of different sequences: these bind to free DNA in the same way that two strands of DNA stick together to make the double helix.

This remarkable technology may offer both the most promising way to early tumour detection and of determining responses to drugs. It also provides a bridge between proteomic and genomic technologies because DNA, captured directly or extracted from isolated cells, can be used for whole genome sequencing. If this system is able to capture cells from most major types of tumour it will indeed provide a rapid route from early detection through genomic analysis to tailored chemotherapy without the requirement for tumour biopsies. In Signs of Resistance we noted that it’s possible to track the response of secondary tumours (metastases) to drug treatment (chemotherapy) using this method of pulling out tumor DNA from blood and sequencing it.

The really optimistic view is that chip isolation of DNA or tumour cells may be a means to cancer detection years, perhaps decades, before any other test would show its presence. By following up with the power of sequencing, the hope is that appropriate drug cocktails can be devised to, so to speak, nip the tumour in the bud.

Wizard’s secret

By the way, Conan Doyle eventually revealed the method behind Sherlock’s wizardry: Watson was a medical man but walked with a military bearing: the skin on his wrists was fair but his face tanned and haggard and he held his left arm in a stiff and unnatural manner. So here was a British army doctor who had served in the tropics (or somewhere equally hot) and been wounded. In 1886 where would that have been? Oh yes, of course. Afghanistan.

Reference

Stott, S.L., Hsu, C.-H., Tsukrov, D.I., Yu, M., Miyamoto, D.T., Waltman, B.A., Rothenberg, M.S., Shah, A.M., Smas, M.E., Korir, G.K., Floyd, Jr., F.P., Gilman, A.J., Lord, J.B., Winokur, D., Springer, S., Irimia, D., Nagrath, S., Sequist, L.V., Lee, R.J., Isselbacher, K.J., Maheswaran, S., Haber, D.A. and Toner, M. (2010). Isolation of circulating tumour cells using a microvortex-generating herringbone-chip. Proceedings of the National Academy of Sciences of the United States of America 107, 18392-18397.