Flipping The Switch

If you spend even a little time thinking about cancer you’ll have realised that it’s very odd – and one oddity in particular may have struck you. A general rule is that it can arise anywhere in the body: breast, bowel and lung are commonly affected, but the more than 200 different types of cancer pop up in lots of other organs (e.g. brain, pancreas), albeit less often. But what about those places of which you hear almost nothing? For example, it’s very unusual to hear of heart or muscle cancers. Which raises the obvious question of why? Is there something going on in these tissues that counters cancer development – acts in some way to slow down tumour formation? And if there is, shouldn’t we find out about it?

Zuzana Keckesova, Robert Weinberg and their colleagues from the Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology and other centres have been scratching their heads over this for a while and they’ve recently published an answer – or, at least, one of the answers.

Getting energy from food

To see how their result fits into the jigsaw puzzle we need a quick recap on the chemical processes that go on in cells to keep them alive, aka, metabolism. Occurring in almost all organisms, glycolysis is a central metabolic pathway in which a series of chemical reactions breaks down sugars into smaller compounds, the energy released being captured as ATP (adenosine triphosphate). Needless to say, it’s complicated – there’s 10 steps and it took the best part of 100 years to work them out completely.

Prising open the black box

The story began with the French obsession with wine (which by now they’ve shared with the rest of the world, bless ’em), specifically why sometimes wine tastes horrible. So they put Louis Pasteur on the case and in 1857 he showed that it was all to do with oxygen: if air (oxygen) is present during the fermentation process the yeast cells will grow but fermentation (i.e. alcohol production) will decrease. This showed that living microorganisms were needed for fermentation and led Eduard Buchner to extract the enzymes from yeast and show that they were sufficient to convert glucose to ethanol (alcohol). In other words, you could do it all in a test tube.

The cartoon shows sugar crossing a cell membrane (a bilayer of phospholipids). The 10 steps of the glycolytic pathway (red dots) convert glucose to pyruvate that can become lactic acid or cross the membrane (another lipid bilayer) of mitochondria. In these ‘cells within cells’ oxygen is consumed to make ATP from pyruvate. Glycolysis yields 2 ATPs from each glucose. In mitochondria ‘aerobic respiration’ produces 38 ATPs per glucose – which is why they have been called the “powerhouse of the cell”. In yeast, fermentation produces alcohol from pyruvate.

This was a stunning achievement because it showed for the first time that living systems weren’t inaccessible black boxes. You could take them to bits, find out what the bits were and reassemble them into something that worked – and that’s really a definition of the science of biochemistry. The upshot was that by the 1930s through the efforts of many gifted scientists, notably Otto Meyerhof and Gustav Embden, we had a step-by-step outline of the pathway now known as glycolysis.

Enter Otto Warburg

But by this point a chap called Otto Warburg had noticed that something odd happened to metabolism in cancer. He showed that tumour cells get most of their energy from glucose using the glycolytic pathway, despite the fact that it is much less efficient than aerobic respiration (2 to 38 ATPs per glucose). And they do this even when lots of oxygen is available. Which seems like molecular madness.

Warburg was part of an amazing scientific galaxy in the period from 1901 to 1940 when one out of every three Nobel Prize winners in medicine and the natural sciences was Austrian or German. Born in Freiburg, he completed a PhD in chemistry at Berlin and then qualified in medicine at the University of Heidelberg. Fighting with the Prussian Horse Guards in the First World War, he won an Iron Cross and followed that up with the 1931 Nobel Prize in Physiology or Medicine for showing that aerobic respiration, that is, oxygen consumption, involves proteins that contain iron. However, he made so many contributions to biochemistry that he was actually nominated three times for the prize.

His discovery about tumour cells led Warburg to suggest, reasonably but wrongly, that faulty mitochondria cause cancers – whereas we now know that it’s the other way around: metabolic perturbation is just one of the consequences of tumour development.

But if upsetting mitochondria gives tumours a helping hand, how about looking for factors that help to keep them normal – i.e. using oxidative phosphorylation. And the obvious place to look is in cells that don’t multiply – i.e. appear cancer-resistant.

Which is the idea that led Keckesova & Co to a ‘eureka’ moment. Searching in muscle cells from humans and mice they discovered a protein, LACTB, lurking in their mitochondria. When they artificially made LACTB in a variety of tumour cells both in vitro and in mice it inhibited their growth. In other words, LACTB appears to be a new ‘tumour suppressor’.

What does it do?

It turns out that LACTB works in a quite subtle way. It’s only found in mitochondria, not in the main body of the cell, and it plays a part in making the membrane that forms the boundary of the “powerhouse of the cell”. Membranes are made of two layers of phospholipids arranged with their fatty tails facing inwards. They work as regulatable barriers via proteins associated with the membrane that control the passage of small molecules – so, for example, pyruvate that we mentioned earlier uses specific proteins to cross the mitochondrial membrane.

But aside from their attached proteins, the lipids themselves are a complex lot: they have a variety of fatty acid tails and different chemical groups decorate the phosphate heads. This gemisch arises in part because the lipids themselves control the proteins that they surround. In other words, if the lipid make-up of a membrane changes so too will the efficiency of embedded transport proteins. LACTB controls the level of one type phospholipid (phosphatidylethanolamine, PE): when LACTB is knocked out more PE is made. Thus this tumour suppressor affects mitochondrial lipid metabolism and hence the make-up of the membrane, and its normal role helps in blocking tumour development.

Layers of lipids with their tails pointing inwards make up cell membranes (left): proteins (red & blue blobs) control what can cross the membrane. Phospholipids themselves are a complex mixture with a variety of head groups and fatty acid tails (right).

And the method behind the madness?

So in this newly-discovered tumour suppressor we have a way in which mitochondria can be subverted to promote tumours by changing the properties of their membrane. But what’s the point? Why might it be more profitable for cancer cells to get most of their energy via a high rate of glycolysis rather than by the much more efficient route of oxidising pyruvate in mitochondria – a switch often called The Warburg effect.

There seem to be two main reasons. One is that pathways branch off from glycolysis that provide components to make new DNA – greater flow though glycolysis makes those pathways more active too – a good thing if cells are going to reproduce. The second is that making abnormal amounts of lactic acid actually helps tumour cells to survive and proliferate, it stimulates the growth of new blood vessels to feed the tumour and it can make the immune response – the  defence normally mounted by the host against tumours – less effective.

By affecting mitochondrial function, mutations that knock out LACTB can give the Warburg effect a helping hand and – if the great man’s still following the literature – he may have noted with some glee that this finding, at least, is consistent with his idea that it all starts in mitochondria!


Keckesova, Z. et al. (2017). LACTB is a tumour suppressor that modulates lipid metabolism and cell state. Nature doi:10.1038/nature21408

And Now There Are Six!!

Scientists eh! What a drag they can be! Forever coming up with new things that the rest of us have to wrap our minds around (or at least feel we should try).

Readers of these pages will know I’m periodically apt to wax rhapsodic about ‘the secret of life’ – the fact that all living things arise from just four different chemical units, A, C, G and T. Well, from now on it seems I’ll need to watch my words – or at least my letters – though maybe for a while I can leave it on the back burner in the “things that have been but not yet” category, to use the melodic prose of Christopher Fry.

Who dunnit?

The problem is down to Floyd Romesberg and his team at the Scripps Research Institute in California.

Building on a lot of earlier work, they’ve made synthetic units that stick together to form pairs – just like A-T and C-G do in double-stranded DNA. But, as these novel chemicals (X & Y) are made in the lab, the bond they form is an unnatural base pair.

Left: Two intertwined strands of DNA are held together in part by hydrogen bonds. Right top: Two such bonds (dotted lines) link adenine (A) to thymine (T); three form between guanine (G) and cytosine (C). These bases attach to sugar units (ribose) and phosphate groups (P) to form DNA chains. Right bottom: Synthetic X and Y units can also stick together and, via ribose and phosphate, become part of DNA.

After much fiddling Romesberg’s group derived E. coli microbes that would take up X and Y when they were fed to the cells as part of their normal growth medium. The cells treat X and Y like the units they make themselves (A, C, G & T) and insert them in new DNA – so a stretch of genetic code may then read: A-C-G-T-X-T-A-C-Y-A-T-… And, once part of DNA, the novel units are passed on to the next generation.

Science fiction?
If this has you thinking creation and exploitation of entirely new life forms?!!’ you’re not alone. Seemingly Romesberg is frequently asked if he’s setting up Jurassic Park but, as he points out, the modified bugs he’s created survive only as long as they’re fed X and Y so if they ‘escape’ (being bugs this would probably be down the drain rather than over a fence), they die. Cunning eh?!!

Is this coming to a gene near you?
No. It is, however, clear that more synthetic bases will be made, expanding the power of the genetic code yet further. What isn’t yet known is what the cells will make of all this. In other words, the whole point of tinkering with DNA is to modify the code to make novel proteins. In the first instance the hope is that these might be useful in disease treatment. Rather longer-term is the notion that new organisms might emerge with specific functions – e.g., bugs that break down plastic waste materials.

At the moment all this is speculation. But what is now fact is amazing enough. After 4,000 million years since the first life-forms emerged, more than five billion different species have appeared (and mostly disappeared) on earth – all based on a genetic code of just four letters.

Now, in a small lab in southern California, Mother Nature has been given an upgrade. It’s going to be fascinating to see what she does with it!


Zhang, Y. et al. (2017). Proceedings of the National Academy of Sciences 114, 1317-1322.

Through the Smokescreen

For many years I was lucky enough to teach in a cancer biology course for third year natural science and medical students. Quite a few of those guys would already be eyeing up research careers and, within just a few months, some might be working on the very topics that came up in lectures. Nothing went down better, therefore, than talking about a nifty new method that had given easy-to-grasp results clearly of direct relevance to cancer.

Three cheers then for Mikhail Denissenko and friends who in 1996 published the first absolutely unequivocal evidence that a chemical in cigarette smoke could directly damage a bit of DNA that provides a major protection against cancer. The compound bound directly to several guanines in the DNA sequence that encodes P53 – the protein often called ‘the guardian of the genome’ – causing mutations. A pity poor old Fritz Lickint wasn’t around for a celebratory drink – it was he, back in the 1930s, that first spotted the link between smoking and lung cancer.

This was absolutely brilliant for showing how proteins switched on genes – and how that switch could be perturbed by mutations – because, just a couple of years earlier, Yunje Cho’s group at the Memorial Sloan-Kettering Cancer Center in New York had made crystals of P53 stuck to DNA and used X-rays to reveal the structure. This showed that six sites (amino acids) in the centre of the P53 protein poked like fingers into the groove of double-stranded DNA.

x-ray-picCentral core of P53 (grey ribbon) binding to the groove in double-stranded DNA (blue). The six amino acids (residues) most commonly mutated in p53 are shown in yellow (from Cho et al., 1994).

So that was how P53 ‘talked’ to DNA to control the expression of specific genes. What could be better then, in a talk on how DNA damage can lead to cancer, than the story of a specific chemical doing nasty things to a gene that encodes perhaps the most revered of anti-cancer proteins?

The only thing baffling the students must have been the tobacco companies insisting, as they continued to do for years, that smoking was good for you.

And twenty-something years on …?

Well, it’s taken a couple of revolutions (scientific, of course!) but in that time we’ve advanced to being able to sequence genomes at a fantastic speed for next to nothing in terms of cost. In that period too more and more data have accumulated showing the pervasive influence of the weed. In particular that not only does it cause cancer in tissues directly exposed to cigarette smoke (lung, oesophagus, larynx, mouth and throat) but it also promotes cancers in places that never see inhaled smoke: kidney, bladder, liver, pancreas, stomach, cervix, colon, rectum and white blood cells (acute myeloid leukemia). However, up until now we’ve had very little idea of what, if anything, these effects have in common in terms of molecular damage.

Applying the power of modern sequencing, Ludmil Alexandrov of the Los Alamos National Lab, along with the Wellcome Trust Sanger Institute’s Michael Stratton and their colleagues have pieced together whole-genome sequences and exome sequences (those are just the DNA that encode proteins – about 1% of the total) of over 5,000 tumours. These covered 17 smoking-associated forms of cancer and permitted comparison of tobacco smokers with never-smokers.

Let’s hear it for consistent science!

The most obvious question then is do the latest results confirm the efforts of Denissenko & Co., now some 20 years old? The latest work found that smoking could increase the mutation load in the form of multiple, distinct ‘mutational signatures’, each contributing to different extents in different cancers. And indeed in lung and larynx tumours they found the guanine-to-thymine base-pair change that Denissenko et al had observed as the result of a specific chemical attaching to DNA.

For lung cancer they concluded that, all told, about 150 mutations accumulate in a given lung cell as a result of smoking a pack of cigarettes a day for a year.

Turning to tissues that are not directly exposed to smoke, things are a bit less clear. In liver and kidney cancers smokers have a bigger load of mutations than non-smokers (as in the lung). However, and somewhat surprisingly, in other smoking-associated cancer types there were no clear differences. And even odder, there was no difference in the methylation of DNA between smokers and non-smokers – that’s the chemical tags that can be added to DNA to tune the process of transforming the genetic code into proteins. Which was strange because we know that such ‘epigenetic’ changes can occur in response to external factors, e.g., diet.

What’s going on?

Not clear beyond the clear fact that tissues directly exposed to smoke accumulate cancer-driving mutations – and the longer the exposure the bigger the burden. For tissues that don’t see smoke its effect must be indirect. A possible way for this to happen would be for smoke to cause mild inflammation that in turn causes chemical signals to be released into the circulation that in turn affect how efficiently cells repair damage to their DNA.


Sir Walt showing off on his return                         to England

Whose fault it is anyway?

So tobacco-promoted cancers still retain some of their molecular mystery as well as presenting an appalling and globally growing problem. These days a popular pastime is to find someone else to blame for anything and everything – and in the case of smoking we all know who the front-runner is. But although Sir Walter Raleigh brought tobacco to Europe (in 1578), it had clearly been in use by American natives long before he turned up and, going in the opposite direction (à la Marco Polo), the Chinese had been at it since at least the early 1500s. To its credit, China had an anti-smoking movement by 1639, during the Ming Dynasty. One of their Emperors decreed that tobacco addicts be executed and the Qing Emperor Kangxi went a step further by beheading anyone who even possessed tobacco.

And paying the price

And paying the price

If you’re thinking maybe we should get a touch more Draconian in our anti-smoking measures, it’s worth pointing out that the Chinese model hasn’t worked out too well so far. China’s currently heading for three million cancer deaths annually. About 400,000 of these are from lung cancer and the smoking trends mean this figure will be 700,000 annual deaths by 2020. The global cancer map is a great way to keep up with the stats of both lung cancer and the rest – though it’s not for those of a nervous disposition!


Denissenko, M.F. et al. ( (1996). Preferential Formation of Benzo[a]pyrene Adducts at Lung Cancer Mutational Hotspots in P53.Science 274, 430–432.

Cho, Y. et al. (1994). Crystal Structure of a p53 Tumor Suppressor-DNA Complex: Understanding Tumorigenic Mutations. Science, 265, 346-355.

Alexandrov, L.D. et al. (2016). Mutational signatures associated with tobacco smoking in human cancer. Science 354, 618-622.

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


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!


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.

Outsourcing the Immune Response

We’re very trendy in these pages, for no other reason than that the idea is to keep up to date with exciting events in cancer biology. Accordingly, we have recently talked quite a lot about the emerging field of cancer immunotherapy – the notion that our in-built immune system will try to kill cancer cells as they emerge, because it ‘sees’ them as being to some extent ‘foreign’, but that when tumours make their presence known it has not been able to do the job completely. The idea of immunotherapy is to give our in-house system a helping hand and we’ve seen some of the approaches in Self Help – Part 2 and Gosh! Wonderful GOSH.

The immune see-saw

Our immune system walks a tight-rope: on the one hand it should attack and eliminate any ‘foreign’ cells it sees (so that we aren’t killed by infections) but, on the other, if it’s too efficient it will start destroying out own cells (which is what happens in auto-immune diseases such as Graves disease (overactive thyroid gland) and rheumatoid arthritis.

Like much of our biology, then, it’s a tug-of-war: to kill or to ignore? And, like the cell cycle that determines whether a cell should grow and divide to make two cells, it’s controlled by the balance between ‘accelerators’ and ‘brakes’. The main targets for anti-tumour immune activity are mutated proteins that appear on the surface of cancer cells – called neo-antigens (see The Shape of Things to Come?)

The aim of immunotherapy then is to boost tumour responses by disabling the ‘brakes’. And it’s had some startling successes with patients going into long-term remission. So the basic idea works but there’s a problem: generally immunotherapy doesn’t work and, so far, in only about one in ten of patients have there been significant effects.

Sub-contracting to soup-up detection

Until now it’s seemed that only a very small fraction of expressed neo-antigens (less than 1%) can turn on an immune response in cancer patients. In an exciting new take on this problem, a team of researchers from the universities of Oslo and Copenhagen have asked: “if someone’s immune cells aren’t up to recognizing and fighting their tumours (i.e. ‘seeing’ neo-antigens), could someone else’s help?” It turns out that many more than 1 in 100 neo-antigens are able to cause an immune response. Even more exciting (and surprising), immune cells (T cells) from healthy donors can react to these neo-antigens and, in vitro at least (i.e. in cells grown in the laboratory), can kill tumour cells.

118. pic

Genetic modification of blood lymphocytes

T cells are isolated from a blood sample and novel genes inserted into their DNA. The engineered T cells are expanded and then infused into the patient. In the latest development T cells from healthy donors are screened for reactivity against neo-antigens expressed in a patient’s melanoma. T cell receptors that  recognise neo-antigens are sequenced and then transferred to the patient’s T cells.

How does that work?

T cells (lymphocytes) circulating in the blood act, in effect, as scouts, scanning the surface of all cells, including cancer cells, for the presence of any protein fragments on their surface that should not be there. The first contact with such foreign protein fragments switches on a process called priming that ultimately enables T cells to kill the aberrant cells (see Invisible Army Rouses Home Guard).

What the Scandinavian group did was to screen healthy individuals for tissue compatibility with a group of cancer patients. They then identified a set of 57 neo-antigens from three melanoma patients and showed that 11 of the 57 could stimulate responses in T cells from the healthy donors (T cells from the patients only reacted to two neo-antigens). Indeed the neo-antigen-specific T cells from healthy donors could kill melanoma cells carrying the corresponding mutated protein.

What can possibly go wrong?

The obvious question is, of course, how come cells from healthy folk have a broader reactivity to neo-antigens than do the cells of melanoma patients? The answer isn’t clear but presumably either cancers can make T cell priming inefficient or T cells become tolerant to tumours (i.e. they see them as ‘self’ rather than ‘non-self’).

And the future?

The more critical question is whether the problem can be short-circuited and Erlend Strønen and friends set about this by showing that T cell receptors in donor cells that recognize neo-antigens can be sequenced and expressed in the T cells of patients. This offers the possibility of a further type of adoptive cell transfer immunotherapy to the one we described in Gosh! Wonderful GOSH.


As one of the authors, Ton Schumacher, put it “Our findings show that the immune response in cancer patients can be strengthened; there is more on the cancer cells that makes them foreign that we can exploit. One way we consider doing this is finding the right donor T cells to match these neo-antigens. The receptor that is used by these donor T-cells can then be used to genetically modify the patient’s own T cells so these will be able to detect the cancer cells.”

And Johanna Olweus commented that “Our study shows that the principle of outsourcing cancer immunity to a donor is sound. However, more work needs to be done before patients can benefit from this discovery. Thus, we need to find ways to enhance the throughput. We are currently exploring high-throughput methods to identify the neo-antigens that the T cells can “see” on the cancer and isolate the responding cells. But the results showing that we can obtain cancer-specific immunity from the blood of healthy individuals are already very promising.”


Strønen, M. Toebes, S. Kelderman, M. M. van Buuren, W. Yang, N. van Rooij, M. Donia, M.-L. Boschen, F. Lund-Johansen, J. Olweus, T. N. Schumacher. Targeting of cancer neoantigens with donor-derived T cell receptor repertoires. Science, 2016.

“Fighting cancer with the help of someone else’s immune cells.” ScienceDaily. ScienceDaily, 19 May 2016.

In the beginning … 

You may have noticed that the American actress Angelina Jolie, who is now employed as a  Special Envoy  for the  United Nations High Commissioner for Refugees, has re-surfaced in the pages of the science media. She first hit the nerdy headlines by announcing in The New York Times that she had had a preventive double mastectomy (in 2013) and a preventive oophorectomy (in 2015).

We described the molecular biology that prompted her actions in A Taxing Inheritance. The essential facts were that she had a family history of breast and ovarian cancer: genetic testing revealed that she carried a mutation in the BRCA1 gene giving her a 87% risk of breast cancer and a 50% chance of getting ovarian cancer.

A star returns

BRCA1 and breast cancer are back in the news as a result of a paper by Jane Visvader, Geoffrey Lindeman and colleagues in Melbourne that asked a very simple question: which type of cell is driven to proliferate abnormally and give rise to a tumour by mutant BRCA1 protein? That is, pre-cancerous breast tissue contains a mixture of cell types: does cancer develop from one in particular –  and, if you blocked proliferation of that type of cell, could you prevent tumours forming?

Simple question but their paper summarises about 10 years of work to come up with a clear answer.

And the villain is …

The mature mammary gland is made up of lots of small sacs (alveoli) lined with cells that produce milk – called luminal cells. Groups of alveoli are known as lobules, linked by ducts that carry milk to the nipple. Most breast cancers start in the lobular or duct cells.

Breast fig copy

Left: Normal breast lobule showing alveoli lined with milk-producing luminal cells connected to duct leading to the nipple. Right: Normal milk sac, non-invasive cancer, invasive cancer.

Things are complicated by there being more than one type of progenitor cell but the Melbourne group were able to show that, in mice carrying mutated BRCA1, one subtype stood out in terms of its cancerous potential. These cells carried a protein on their surface called RANK (which is member of the tumour necrosis factor family). They had gross defects in their DNA repair systems (so they can’t fix genetic damage) and they’re highly proliferative. Luminal progenitors that don’t express RANK behave normally.

Slide1 copy

Scheme representing normal and abnormal cell development. The basic idea is that different types of cells evolve from a common ancestor. The Australian work identified one type of luminal progenitor cell that carries a protein called RANK on its surface (pink cell) as being a prime source of tumours. RANK+ cells have defective DNA repair systems so they accumulate mutations (red cells) more rapidly than normal cells, a feature of tumour cells.

In mice with mutant BRCA1 a monoclonal antibody (denosumab) that blocks RANK signalling markedly slowed tumour development. In a small pilot study blockade of RANK inhibited cell proliferation in breast tissue from human BRCA1-mutation carriers.


How effective blocking the activation of RANK signalling will be in preventing breast cancer is anyone’s guess but the idea behind the work of the Australian group cannot be faulted. Being able to prevent the ‘starter’ cells from launching themselves on the pathway to cancer driven by mutation in BRCA1 would mean that women in Angelina Jolie’s position would not have to contemplate the drastic course of surgery. The question is: will the preliminary mouse results lead to something that works in humans and, moreover, does so with high efficiency. As ever in cancer, watch this space – but don’t hold your breath!


Nolan, E. et al. (2016). RANK ligand as a potential target for breast cancer prevention in BRCA1-mutation carriers.


New Era … Or Déjà vu?


Readers who follow events in the US of A – beyond the bizarre unfolding of the selection of the Republican Party’s nominee for President of the United States – may have noticed that the presidential incumbent put forward another of his bright ideas in the 2016 State of the Union Address. The plan launched by President Obama is to eliminate cancer and to this end $1 billion is to go into a national initiative with a strong focus on earlier detection, immunotherapy and drug combinations. It’s called a Moonshot’, presumably as a nod to President Kennedy’s 1961 statement that America should land a man on the moon (and bring him back!).011316_SOTU_THUMB_LARGE

A key aim of Moonshot is to improve all-round collaboration and to ‘bring about a decade’s worth of advances in five years.’ Part of this involvesbreaking down silos’ – which apparently is business-speak (and therefore a new one on me) for dealing with the problem of folk not wanting to share things with others in the same line of work. So someone’s spotted that science and medicine are not immune to this frailty.silo_mentality

On the home front …

In fact the President could be said to be slightly off the pace as, in October 2015, Cancer Research UK launched ‘Grand Challenges’ – a more modest (£100M) drive to tackle the most important questions in cancer. They’ve pinpointed seven problems and, helpfully, six of these will not be new to dedicated readers of these pages. They are:

  1. To develop vaccines (i.e. immunotherapy) to prevent non-viral cancers;
  2. To eradicate the 200,000 cancers caused each year by the Epstein Barr Virus;
  3. To understand the mutation patterns caused by different cancer-causing events;
  4. To improve early detection;
  5. To map the complexity of tumours at the molecular and cellular level;
  6. To find a way of targetting the cancer super-controller MYC;
  7. To work out how to target anti-cancer drugs to specific cells in the body.

{No/. 2 is the odd one out so it clearly hasn’t been too high a priority for me but we did talk about Epstein Barr Virus in Betrayed by Nature – phew!}.

But wait a minute

Readers of a certain age may be thinking this all sounds a bit familiar and, of course, they’re right. It was in 1971 that President Richard Nixon launched the ‘war on cancer’, the aim of which was to, er, to eliminate cancers. Given that 45 years on in the USA there’ll be more than 1.6 million new cases of cancer and 600,000 cancer deaths this year, it’s tempting to conclude that all we’ve learned is that things are a lot more complicated than we ever imagined.

Well, you can say that again. Of the several hundred genes that we now know can play a role in cancers, two are massively important MYC (‘mick’) and P53. Screen the scientific literature for research publications with one of those names in the title and you get, wait for it, over 50,000 for ‘MYC’ and for P53 over 168,000. It’s impossible to grasp how many hours of global sweat and toil went into churning out that amount of work – and that’s studies of just two bits of the jigsaw!

So 45 years of digging have yielded astonishing detail of the cellular and molecular biology – and that basis will prove essential to any rational approach to therapy. It’s a slow business this learning to walk before you run! But we can be rather more up-beat. Alongside all the science there have come considerable improvements in treatments. Thirty years ago one in four of those diagnosed with a cancer survived for more than 10 years. Now it’s almost one in two. But it’s a hugely variable picture: for breast cancer the 10 year overall survival rate is nearly 80% and for testicular cancer it’s over 98%. However, for lung cancer and cancer rates remain below 5% 1%, respectively. For these and other cancers there has been very little progress.

So 45 years of digging away have yielded astonishing detail of the cellular and molecular biology – and that basis will prove essential to any rational approach to therapy. It’s a slow business this learning to walk before you run! But we can be rather more up-beat. Alongside all the science there have come considerable improvements in treatments. Thirty years ago one in four of those diagnosed with a cancer survived for more than 10 years. Now it’s almost one in two. But it’s a hugely variable picture: for breast cancer the 10 year overall survival rate is nearly 80% and for testicular cancer it’s over 98%. However, for lung cancer and pancreatic cancer rates remain below 5% and 1%, respectively. For these and other cancers there has been very little progress.

All systems go?

Well, maybe. Moonshot is aimed at better and earlier diagnosis, more precise surgery and radiotherapy, and more drugs that can be better targeted. Oh, and bearing in mind that one in three cancers could be prevented, keeping plugging away at lifestyle factors.

How will it fare? Well, now we’re in the genomic era we can be sure that the facts mountain resulting from 45 years of collective toil will be as a molehill to the Everest of data now being mined and analysed. From that will emerge, we can assume with some confidence, a gradual refinement of the factors that are critical in determining the most effective treatment for an individual cancer.

Just recently we described in The Shape of Things to Come the astonishingly detailed picture that can be drawn of an individual tumour when it’s subjected to the full technological barrage now available. As we learn more about the critical factors, immunotherapy regimens will become more precise and the current response rate of about 10% of patients will rise.

Progress will still be slow, as we noted in The Shape of Things to Come – don’t expect miracles but, with lots of money, things will get better.

The Shape of Things to Come?

One of the problems of trying to keep up with cancer – and indeed helping others to do so – is that you (i.e. ‘I’) get really irritated with the gentlemen and ladies of the press for going over the top in their efforts to cover science. I have therefore been forced to have a few rants about this in the past – actually, when I came to take stock, even I was a bit shocked at how many. Heading the field were Not Another Great Cancer Breakthough, Put A Cap On It and Gentlemen… For Goodness Sake. And not all of these were provoked by The Daily Telegraph!

If any of the responsible reporters read this blog they probably write me off as auditioning for the Grumpy Old Men tv series. But at least one authoritative voice says I’m really very sane and balanced (OK, it’s mine). Evidence? The other day I spotted the dreaded G word (groundbreaking) closely juxtaposed to poor old Achilles’ heel – and yes, it was in the Telegraph – but, when I got round to reading the paper, I had to admit that the work referred to was pretty stunning. Although, let’s be clear, such verbiage should still be banned.

A Tumour Tour de Force

The paper concerned was published in the leading journal Science by Nicholas McGranahan, Charles Swanton and colleagues from University College London and Cancer Research UK. It described a remarkable concentration of current molecular fire-power to dissect the fine detail of what’s going on in solid tumours. They focused on lung cancers and the key steps used to paint the picture were as follows:

1. DNA sequencing to identify mutations that produced new proteins in tumour cells (called tumour-associated antigens or ‘neoantigens’ – meaning molecular flags on the cell surface that can provoke an immune response – i.e. the host makes antibody proteins that react with (stick to) the antigens). Typically they found just over 300 of these ‘neoantigens’ per tumour – a reflection of the genetic mayhem that occurs in cancer.

2 tumoursVariation in neoantigen profile between two multi-region sequenced non-small cell lung tumours. There were approximately 400 (left) and 300 (right) neoantigens/tumour

  • Blue: proportion of clonal neoantigens found in every tumour region.
  • Yellow: subclonal neoantigens shared in multiple but not all tumour regions.
  • Red: subclonal (‘private’) neoantigens found in only one tumour region.
  • The left hand tumour (mostly blue, thus highly clonal) responded well to immunotherapy (from McGranahan et al. 2016).

2. Screening the set of genes that regulate the immune system – that is, make proteins that detect which cells belong to our body and which are ‘foreign.’ This is the human leukocyte antigen (HLA) system that is used to match donors for transplants – called HLA typing.

3. Isolating specialised immune cells (T lymphocytes) from samples of two patients with lung cancer, growing them in the lab to expand the population and testing how good these tumour-infiltrating cells were at recognizing the abnormal proteins (neo-antigens) on cancer cells.

4. Detecting proteins released by different types of infiltrating T cells that regulate the immune response. These include so-called immune checkpoint molecules that limit the extent of the immune response. This showed that T cell subsets that were very good at recognizing neo-antigens – and thus killing cancer cells (they’re CD8+ T cells or ‘killer’ T cells) also made high levels of proteins that restrain the immune response (e.g., PD-1).

5. Showing that immunotherapy (using the antibody pembrolizumab that reacts with PD-1) could significantly extend survival of patients with advanced non-small cell lung cancer. We’ve already met this approach in Self-help Part 1.

The critical finding was that the complexity of the tumour (called the clonal architecture) determines the outcome. Durable benefit from this immunotherapy requires a high level of mutation but a restricted range of neo-antigens. Put another way, tumours that are highly clonal respond best because they have common molecular flags present on every tumour cell.

6. Using the same methods on some skin cancers (melanomas) with similar results.

What did this astonishing assembly of results tell us?

It’s the most detailed picture yet of what’s going on in individual cancers. As one of the authors, Charles Swanton, remarked “This is exciting. This opens up a way to look at individual patients’ tumours and profile all the antigen variations to figure out the best ways for treatments to work. This takes personalised medicine to its absolute limit where each patient would have a unique, bespoke treatment.”

He might have added that it’s going to take a bit of time and a lot of money. But as a demonstration of 21st century medical science it’s an absolute cracker!


McGranahan et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 10.1126/science.aaf490 (2016).


Transparently Obvious


Scientists have a well-earned reputation for doing odd things – by which I mean coming up with a ‘finding’ that leaves me, at least, wondering how, in the name of all things wonderful, they ever got money to do their study. To be fair, it’s the ‘social scientists’ – rather than the ‘real’ lot – that excel in this field. An example? Take your pick. They crop up pretty well weekly in the press. I liked the one on how something called ‘personal congruence’ affects marriage survival. The more congruence you and your partner have the better your chances: if, over time, your congruence goes down the tubes, your relationship will surely follow. But what on earth is congruence? Seemingly it’s a ‘state of agreeing.’ Lots of it equals harmony, loss of it = discord. So, it is what you remember from school geometry: it means more or less equal. Wow! Now I’ve grasped the upshot of this ‘study’: agreeably happy couples tend to make it: pairings based on whacking each other with frying pans tend to end in tears. Why didn’t they tell us earlier!!



Fortunately, in my world, even the weirdies usually turn out to be quite sensible, once you know what’s going on. Many moons ago a girl-friend asked me if I’d like to see her collection of axolotls. Not having a clue what she was on about I gave it an excited ‘yes please’. Whilst it mayn’t have been what I was hoping for (I was very young back then), I immediately fell in love with these wonderful amphibians that I’d never heard of as she explained what I should have known: these ‘Mexican walking fish’ have very large embryos which makes them particularly useful for studying development. These sensational salamanders really are amazing, not least because they can regenerate entire limbs after they’ve been chopped off.

More recently there’s been another unlikely recruit to the scientific armoury: the zebrafish – a tropical freshwater fish from the Himalayas. This mighty minnow was the first vertebrate to be cloned which led to its being genetically modified to give a transparent variety. That’s all good fun but what on earth is the point of a see-through fish? Well, in Betrayed by Nature we pointed out that you can actually watch tumours growing in transparent zebrafish and we got so excited by that we even included a photo – kindly provided by Richard White of the Dana Farber Cancer Institute in Boston. The cancer was a melanoma which had grown into a black mass about 1 cm in diameter in the fish’s body after a small number of tumour cells had been injected a couple of weeks earlier.

And the driver is …

Nearly 15 years ago, just as the first complete sequence of human DNA was being unveiled, Mike Stratton and his colleagues at the Sanger Centre in Cambridge discovered a mutation that arises in about two-thirds of all malignant melanomas. It’s in a gene called BRAF. The protein made by the gene is an enzyme that’s part of a signalling pathway that pushes cells to divide. The mutation changes the shape of BRAF protein so it works 24/7 as an enzyme: the pathway is no longer controlled by a message from the world beyond the cell. It’s a ‘molecular switch’ that’s been flipped by mutation to act as a cancer ‘driver.’

Richard White and his colleagues showed that the same mutation drove melanoma development in zebrafish and that when it did so something remarkable happened. As the tumours got going they turned on a gene that is normally only required during the first 72 hours after fertilization. The gene’s called crestin – because it’s switched on in a tissue called the neural crest where crestin protein helps to form the bony support for the gills. After that it’s switched off and crestin protein never appears again. Except in the pigment-containing cells called melanocytes when they are turning into a tumour.

Seeing the problem

In a great example of how science can work, Charles Kaufman, Leonard Zon and colleagues in Boston and other centres took this finding and made another transgenic variant of the transparent zebrafish. They cut out the stretch of DNA that controls whether the crestin gene is ‘on’ or ‘off’ and hooked it up to a gene that makes a green fluorescent protein (GFP). Result: when the machinery of a cell turns crestin on, GFP is also made – and the cell glows green under the appropriate light. Hence you would expect to see a glowing neural crest early in development but thereafter a non-glowing fish. Unless it has a melanoma. And Zon & Co saw exactly that. Because green fluorescent protein glows so brightly, a single cell shows up and it turned out that whenever one green cell was detected it always went on to expand and grow into a large melanoma tumour.

1 cell to mel

Tracking a single cell turning into a tumour over 6, 9, 11.5 and 17 weeks. The green fluorescence marks an early developmental gene (crestin) being re-activated in a melanoma tumour (from Kaufman et al., 2016).

But why might it be useful to ‘see’ single cells?

Since the original finding by Stratton & Co more detailed studies have confirmed that mutated BRAF is indeed an important ‘driver’ in about two-thirds of malignant melanoma. But here’s the odd thing: lots of melanocytes (the cells that can turn into melanomas) have mutated BRAF – but they don’t become cancerous. Why not? And there’s something else: it’s well-known that ultraviolet radiation in sunlight causes many melanomas and they do indeed often arise on exposed skin – but they can also crop up in places where, as they say, the sun doesn’t shine. So clearly, important though mutated BRAF and sunlight are, there’s something else that’s critical for malignant melanoma.

The Kaufman experiment was remarkable, not least because it offers a way of getting at this key question of what happens in a cell to kick it off as a tumour, by comparison with a near neighbour that remains ‘normal.’

The tumour cells used in this model carry mutated BRAF and another gene, P53, was knocked out. This gives two major genetic drivers and it may be that further genetic changes aren’t needed. If that’s the case, then the decisive push must come either from epigenetic changes (that affect gene expression without change in DNA sequence) or from adaptations of the tumour microenvironment to provide an optimal niche for expansion. At the moment we don’t know very much about these critical areas of cancer biology. Being able to follow single cells may lead us to the answers.

Keep your eye on the transparent minnows!


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


Open Wide for Pasty’s Throat


Once upon a time (1903 to be exact) a very rich Adelaide family acquired a new member in the form of a little boy whom they christened Norman. Most of the family were doctors and, so well-heeled were they, when young Norm reached the age of 10 they clubbed together and sent him off to the Old Country – and not just to any bit of Merrie England but to Eton (the school, of course, not the rustic parish, generally held to be the most expensive of all – fees currently about £36,000 a year, not counting extras). Norm never returned: South Australia’s loss was Britain’s gain.

We all know what happens to kids that go to Eton – but our Adelaide man was different. For one thing he was very bright and for another he had his family’s love of medicine. He ended up specializing in the thorax – the bit between the neck and the tummy that includes the oesophagus, commonly known as the foodpipe or gullet. Eton probably helped get him started but, even more usefully, some bright spark there gave him the nickname ‘Pasty’ – so great an improvement on Norman that it stuck for life. ‘Pasty’ Barrett ended up as a consultant at St. Thomas’ Hospital where, in 1947, he successfully repaired a ruptured oesophagus – a surgical first for a hitherto fatal condition.

Shortly after that, in 1950, he described finding that sometimes the cells lining the gullet change in appearance, switching from multiple layers of flat cells to a single layer of cells that look like those found in the intestine. We know now that this change is caused by acid from the stomach being squeezed up into the oesophagus. Occasional regurgitation is called heartburn but when it’s persistent it becomes gastric reflux disease – and in about 10% of those cases sustained irritation caused by the stomach juices upsets the cells lining the gullet and they undergo the change to what is now called Barrett’s oesophagus.

Who cares about Barrett’s?

Well, we should all at least take note because a few percent of those with Barrett’s oesophagus will get cancer of the oesophagus, which is now the sixth most common cause of cancer-related death world-wide. Oesophageal cancer has become more common over the last 40 years, men are more prone to it than women and it kills about 15,000 people in the USA each year and nearly 8,000 in the UK. It’s very bad news. Most cases aren’t discovered until the disease has spread and it is then more or less untreatable. The prognosis is dismal: the five-year survival figure is barely 15%. Part of the problem is that the main sign is pain or difficulty in swallowing, often ignored until it is too late.

For many years the only way of finding abnormal tissue was by an endoscopy – a tube with a camera pushed down the throat – both unpleasant and expensive. There has, therefore, been a desperate need for an easy, cheap, non-invasive test to screen for Barrett’s oesophagus.

Professor Rebecca Fitzgerald

     Professor Rebecca             Fitzgerald

Pill on a string

Enter Rebecca Fitzgerald, a member of the Department of Oncology in Cambridge and a consultant at Addenbrooke’s Hospital, with a brilliantly simple development from earlier attempts to screen the lining of the gullet. The patient swallows a kind of tea-bag on a string which is then pulled up from the stomach. The ‘tea-bag’ is actually a capsule about the size of a multi-vitamin pill containing a sort of honeycomb sponge covered with a coating that dissolves in a few minutes when it reaches the stomach. As the sponge comes up it picks up cells from the gullet lining (about half a million of them) that can then be analysed. The whole gizmo’s called a ‘Cytosponge’. It works with no problems and because it collects cells from the length of the gullet it gives a complete picture, rather than the local regions sampled in biopsies.

Pill on a string

                 Pill on a string

Cytosponge (left) and being drawn up the gullet (right)

       Cytosponge (left) and being             drawn up the gullet (right)






What we’ve learned

The hope was that the cells picked up by Cytosponge could be sequenced – i.e. their DNA code could be obtained – and that this would reveal the stages of oesophageal cancer development and hence whether a given case of Barrett’s would or would not progress to cancer. The phases of Barrett’s oesophagus involve a change in the shape of cells lining the tube (from thin, flat cells called squamous epithelial cells to taller columnar cells resembling those in the intestine). This change is called metaplasia: the abnormal cells may then proliferate (dysplasia). If this stage can be detected it’s possible to remove the abnormal tissue by using endoscopic therapy before the condition progresses to full carcinoma.

Remarkably, whole-genome sequences from Barrett’s and from oesophageal carcinoma showed that multiple mutations (changes in DNA sequence) accumulate even in cells that are over-proliferating but look normal. The picture is similar to the ‘battlefield of hundreds of competing mutant clones’ in normal eyelid skin that we saw in The Blink of an Eye.

As the condition progresses the range of mutations increases: in particular, regions of DNA are duplicated – so that the genes therein are present in abnormal numbers. Typically there were 12,000 mutations per person with Barrett’s oesophagus without cancer and 18,000 mutations within the cancer.

Even from this mayhem there emerged mutation patterns (changes in the letters of the DNA code, e.g., A to a G or C to a T) characteristic of the damage caused to the cells lining the oesophagus by splashing stomach acid. These ‘fingerprints’ were found in both Barrett’s and oesophageal cancer – consistent with them being very early events – parallelling the specific mutations in lung cancer caused by tobacco carcinogens.

But …

The great hope was that the spectrum of mutations would identify precursors to cancer and hence those patients requiring treatment. In fact these horribly heterogeneous tissues – a real genetic gemisch – show surprisingly little mutational overlap between Barrett’s oesophagus and oesophageal cancer.

However, it’s possible to take the cells collected by the Cytosponge and screen them for the presence of specific proteins (using antibodies) and it turns out that one in particular, TFF3 (Trefoil Factor 3), provides a highly accurate diagnosis of Barrett’s oesophagus. In addition, although the genetic changes that occur during the progression from Barrett’s to cancer are complex, mutations in one gene (P53 – the ‘guardian of the genome’) are common in pre-cancerous, high grade dysplasia and thus provide an indicator of risk.

All of which means that we haven’t ‘conquered’ oesophageal cancer – but thanks to these remarkable advances we have a much better understanding of its molecular basis. Even more importantly, it’s possible to detect the early stages – and do something about it.

AND … whilst making a major contribution to all this, Rebecca Fitzgerald very kindly found time to make suggestions and provide additional information for this piece.


Ross-Innes, C.S., Fitzgerald, R.C. et al. (2015). Nature Genetics 47, 1038-1046.