The Blame Game

“Why do people get it?” — perhaps the most frequently asked question about cancer. But nowadays most of us can come up with a quick answer: “It’s mutations” — that is, damage to DNA, our genetic material. Such changes are most commonly to the sequence of DNA (alterations to individual bases or loss or gain of bits). That’s a molecular biologist’s answer. What we really want to know is “Why?” How do these changes come about and, of course, what can we do about them?

The things we do …

We’ve known part of the answer for a long time — it’s what we do to ourselves, stupid! The best known example is smoking, shown to cause lung cancer in the 1930s. We know now that chemicals in cigarette smoke damage DNA and they’re so good at it that over 90% of lung cancer is down to smoking. So pernicious is the habit that it killed about 100 million people in the twentieth century and, unless something pretty drastic happens, the number of deaths this century will be one billion.

It’s true, we have made some progress. Most countries now regulate smoking. Bhutan (bless ’em) was the first nation to outlaw smoking in all public places. In the UK tobacco advertising is banned, as is smoking in all work places. But none of this happened until we’d got well into the twenty-first century! Oh, and if you’re wondering how the country that likes to style itself the world leader is getting on, Congress has so far managed to avoid passing any nationwide smoking ban and left it to individual states — with the result that across the USA laws range from total bans to no regulation of smoking at all! All told, the saga has been one of the more staggering examples of political impotence.

I’m sure you can think of other daft things we do to propel us to our cancer graves but we need to move on by noting that, aside from what we do to ourselves, the world we live in contributes something of a helping hand. Thus some useful foods nevertheless contain harmful substances and even the ground we stand on gives off low levels of radiation. And there’s not a lot we can do about such things.

… And are done to us

Then there’s heredity — the state of our DNA when we get it. It’s been clear for some time that mutations passed to us at birth kick off about 10% of all cancers (see for example, A Taxing Inheritance).

Way back in 1866 Paul Broca suggested it might be possible to inherit breast cancer. He’d looked at his wife’s family tree and noted that ten out of twenty-four women, spread over four generations, had died from that disease and that there had been cases of other types of cancer in the family as well. This large proportion was not, he believed, mere chance. Now we know that a changed (mutated) form of a gene (a unit of heredity), passed from generation to generation, was almost certainly responsible for the suffering of this family.

So broadly speaking there are two long-recognized categories that cause cancer —‘environmental’ and ‘hereditary’ — and, although we cheat by lumping things that we can control (e.g., smoking, eating too much red meat and sunbathing) into the ‘environmental’ camp (there should be a separate group: ‘stupidity’), many factors really are beyond our control.

As ever, it’s worse than that

Lurking in the wings for many years now has been a potential third cause that arises from a slightly tricky concept — namely the fact that our DNA, the genetic rock upon which all life is built, isn’t rock-like at all. In fact the chemistry of DNA makes it inherently unstable. Thinking about it from the viewpoint of evolution, of course it’s unstable: it has to be to permit change as new genes, and hence new proteins, are made and unmade — allowing life forms to advance. Think of it like close relationships: we’re fond of calling such things ‘permanent’, ‘unchanging’, ‘solid as a rock’ even. But they’re not: they change all the time, adapting to our shortcomings and to how individuals develop and mature.

With that in mind maybe it’s less surprising to find that DNA reacts with a wide range of chemicals, some that we consume but others arising from the natural reactions of the body — products of metabolism in fact. And then, speaking of shortcomings, there’s the truism that ‘nobody’s perfect’ and the realization that this applies to the mechanics of DNA replication as well as everything else. In others words, every time we make a new cell its DNA differs from the original. Cells have remarkably smart methods for correcting most mistakes made during replication but, inevitably, some get through and become fixed in the new genome.

Although ‘replicative mutations’ have been known for a while, nobody had come up with a way of measuring how much they contribute to cancers. Step forward Bert Vogelstein and Cristian Tomasetti at Johns Hopkins University with the idea of looking at ‘stem cells’ — cells that can divide to make more of themselves or to turn themselves into specialized cell types. They reasoned, bearing in mind that with every division there’s a risk of a cancer-causing mutation in a daughter cell, that if you knew the number of stem cells in an organ and you could estimate the total number of divisions over a lifetime, that might relate to cancer risk.

Indeed it did. In spades, because it turned out to account for two-thirds of all cancers. In other words, the majority of cancers arise because of cumulative mutations caused by internal agents.

Quick test to see if that fits with something we know: cancers of the intestine. Cancers of the duodenum (the first section of the small intestine) are rare compared with those of the colon (the large intestine, into which the duodenum empties). For 2017 in the USA the estimates are 1390 and 50,260 deaths, respectively – that’s about 0.2% of all cancer deaths versus 8% for colon cancers. Sure enough, Tomasetti and Vogelstein estimated the cell division rate to be about 100 times greater in the colon over a lifetime.

Mice, somewhat curiously, are the other way round: they pick up more cell divisions in their small intestine — and more cancers — than in their colon.

This correlation of rising risk with increasing number of divisions held over 31 different types of cancer — as it did when extended from USA to world-wide data, thereby eliminating a bias from environmental factors.

The upshot of this is that to ‘environmental’ and ‘hereditary’ factors we need to add a third category of ‘replication errors.’

Real-life examples of the impact of replicative mutations on lung cancer and prostate cancer.

About 90% of lung cancers are preventable: heredity plays no significant role and the estimate is that 35% of all driver (i.e. cancer-promoting) mutations are due to replication errors. For prostate cancers there is no evidence that environmental factors are significant and hereditary factors account for 5 to 9% of cases. The remaining 95% of driver gene mutations are estimated to be replication errors. None of these cancers are preventable. Clouds represent contributions from environmental factors. Gray dots: Environmental mutations, Yellow dots: Replicative mutations, Blue dots + H: Hereditary mutations (from Tomasetti et al. 2017).

 Causes of driver mutations in 18 types of female cancer (UK).

The colour codes are the same for hereditary (left), replicative (centre), and environmental (right) factors and are from white (0%) to brightest red (100%).

The left-hand schematic indicates that inherited mutations are not statistically significant in these cancers (note that Paul Broca’s findings related to fewer than 10% of breast cancers – the proportion we now know to be caused by abnormal genes passed from parent to child).

B, brain; Bl, bladder; Br, breast; C, cervical; CR, colorectal; E, esophagus; HN, head and neck; K, kidney; Li, liver; Lk, leukemia; Lu, lung; M, melanoma; NHL, non-Hodgkin lymphoma; O, ovarian; P, pancreas; S, stomach; Th, thyroid; U, uterus (from Tomasetti et al. 2017).

Controversial or what …?

It’s fair to say that the estimate of two-thirds of all cancers being down to internal faults was a surprise to many.

It has to be said that there’s a continuing debate about the precise numbers — not least because figures for cell divisions in some tissues aren’t available and also because of somewhat vaguer problems, e.g., to what extent to external assaults contribute to replication errors.

Nevertheless, it now seems clear that what Tomasetti and Vogelstein call “bad luck” can be blamed for a significant number of cancers. That’s good because knowing that it’s not your ‘fault’ may help some patients but we need to be wary of promoting that message too strongly in the media.

The fact is that, whatever the proportion might be that we can put down to “bad luck”, there are still a great many cancers that can be prevented.

What’s to be done?

Now we know what to blame we can return to the question of what can be done. It won’t take long because at the moment the answer is ‘not much’. The accumulation of mistakes from replication errors is random, so we cannot predict who will find themselves with a critical (i.e. cancer-producing) set. But that scarcely matters as we have no way of preventing them happening. So all we can do at the moment is deal with what presents itself with the treatments currently available, comforting ourselves that in the long-term things like gene–editing might enable us to rectify critical replication mutations.

So, like a lot of fascinating advances in the cancer field, the take-home message here is “that’s all very interesting but in the meantime we need to keep focusing on the possible: the fact that if we stopped smoking, got people to eat sensibly and gave everyone decent sanitation we could cut cancers by half”. Give or take a few percent!

References

Tomasetti, C. et al., (2017). Stem cell divisions, somatic mutations, cancer etiology, and cancer prevention. Science 355, 1330–1334.

Tomasetti, C. and Vogelstein, B. (2015). Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science 347, 78-81.

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Hares And Tortoises

You may have noticed that the last few months have seen a bit of a DNA-fest in these pages. Don’t blame me. It’s all the fault of them scientists beavering away in their labs. We’ve just done “Making Movies in DNA“, in “And Now There Are Six!!” the genetic code was expanded from four to six units by making two new ones artificially and in “How Does DNA Do It?” we saw how words can be transformed into a sequence of DNA.

Now they’re at it again – or at least Stephen Kowalczykowski, James Graham and colleagues of the University of California at Davis are – revealing yet more astonishing things about this molecule, just when you could be thinking we’ve got the hang of it.

I might add that I’m grateful to my correspondent David Archer of The Society of Biology for bringing this piece of work to my notice as I’d missed it in the journal Cell (cries of ‘shame’ and ‘shurely shome mistake’ mingle in the background).

What is it this time?  

Well it’s two really astonishing things about DNA replication – the process by which double-stranded DNA is pulled apart so that each strand can act as a template for making a new DNA molecule. Result: as cells progress towards division, they double their DNA content so that equal amounts can be given to each new daughter cell. The first source of amazement is that Stephen K & chums have filmed this happening in real time. That’s a terrific feat – but what it reveals is quite bizarre.

Up to now it’s been assumed that the protein machines (DNA polymerases) doing the biz trundle along each of the separated strands of parental DNA at more or less the same speed. It would seem to make no sense to do otherwise and risk ending up with the job half done. In other words, the duplication of the two strands is coordinated. Is that what K & Co found? Not a bit of it! Extraordinary to relate, it appears that there’s no coordination between the strands at all!! Not for the first time in the history of molecular biology a technical advance has thrown up the totally unexpected. Before we look at the results in a bit more detail, a little background might be useful.

One divides into two

Making two identical copies of DNA from one original happens every time one cell divides to make two. And there’s a lot of it about. As is well known, we all start out as one cell (i.e. a fertilized egg) that turns into a human being – 50 trillion cells (that’s 5 + 13 zeroes). And even after we’ve been assembled it takes a lot of cell-making to keep us ticking over – about one million new cells every second. Just take a second to think about that: DNA comes in the well-known form of a double helix – two strands made up of chemical units (called nucleotides) linked together. Each unit has one of four bases (cytosine (C), guanine (G), adenine (A), or thymine (T)) and the strands are “complementary” because C pairs with G and A with T – a rigid rule that means if you know the sequence of bases in one strand you can work out what it is in the other. So far so simple. But, as we noted in “How Does DNA Do It?”, the coding power of DNA lies in its size. In us three billion letters are available to do the encoding. That is, there are just over 3,000 million units in each chain – i.e. 3,000 million base-pairs all told. And all of these are copied (twice) for every new cell.

DNA replication: The double helix is ‘unzipped’ so that each separated strand (turquoise) can act as a template for replicating a new partner strand (green). This creates a ‘replication fork’ – two branches of single stranded DNA. The new strands are made by protein complexes called DNA polymerases chugging along the parent strands, making new, complementary, strands as they go. There’s a small technical wrinkle here: new DNA chains can only be extended in one direction. This means that, while one strand can be made continuously (the leading strand), the other has to be put together in short bits as the parent strand is unwound, with the bits being joined up afterwards (the lagging strand).

 

 

Timing is everything

So the cell’s task is to unzip the double helix and use each exposed strand as a template for building a new partner strand. Things are helped by DNA being split into fragments (chromosomes: 23 pairs in humans + 2 sex chromosomes, 46 per cell all told). Even so, chromosomes are huge: the longest (chromosome 1) has nearly 250 million base-pairs; the shortest (chr 21) has about 47 million. The problem for the machinery that has evolved for the job is that it cranks along at 50 pairs per second – roughly a month per chromosome. But in a normal cell cycle the whole business is done in about two hours! That’s made possible because replication doesn’t do the obvious: start at one end and work its way to the other. Cunningly it hits lots of ‘start points’ – up to 100,000 in a single cell – making lots of short bits at the same time that are then joined up. In other words replication proceeds simultaneously from many different sites in chromosomes. Enzymes join the pieces together to make the final, complete copy.

It’s rather like you having some horribly repetitive chore to do – washing up after a big dinner. On your own you might start at one end of the pile and work through it but, far better, get one member of the family to do the plates, another the cutlery, etc. and – job done!!

Now for today’s bit of amazing science

What Kowalczykowski and friends did was to extract DNA from bugs (E.coli bacteria in fact, that can make DNA about 20 times faster than human cells), set up a replication system and measure what went on by microscopy, using a dye (SYTOX Orange, which is fluorescent) that sticks to complete double helices but not to single strands. Thus they could track progress along a strand as a new double helix formed. What they saw was that each strand acted independently of the other. Overall, the rate of replication of the two strands was about the same (as it must be in the end) but along the way there were stops and starts and sometimes one strand would grow at ten times the speed of the other. How weird is that?!!

Seeing DNA being made. In this picture microscopy reveals three extending stretches of double-stranded DNA being made (Graham et al. 2017). Click here to see video.

You could picture DNA replication as one of those Swiss railway trains cranking up a mountain at an improbable angle, using a rack-and-pinion to stop it sliding backwards. Think of the engaging cogs as new base-pairs. The train just keeps chugging along until it reaches the its next stop. But why doesn’t the DNA-making machinery do the same? Well, we haven’t much of a clue. One difference is that the train has its track (and rack) laid out before it, whereas DNA is continuously being unwound to open the template. Some bits are more difficult to unwind than others and this variation may cause the system to go in fits and starts. Another contribution many come from the many proteins involved in this complicated process. As well as the polymerases there are things that unwind DNA, stabilize it, stitch new bits together, etc. and these complexes are continuously forming, falling apart and re-assembling – all of which gives plenty of scope for erratic behaviour.

Fact of the matter is, we don’t know. So, in revealing completely unexpected behaviour, this technical triumph throws up the question of how two strands working independently manage, in the end, to come up with the perfect finished product.

But hey! This wouldn’t be science if we had all the answers!

Reference

Graham, J.E., Marians, K.J., Kowalczykowski, S.C. (2017). Independent and Stochastic Action of DNA Polymerases in the Replisome. Cell 169, 1201–1213.

Making Movies in DNA

Last time we reminded ourselves of one of the ways in which cancer is odd but, of course, underpinning not just cancers but all the peculiarities of life is DNA. The enduring wonder is how something so basically simple – just four slightly different chemical groups (OK, they are bases!) – can form the genetic material (the instruction book, if you like) for all life on earth. The answer, as almost everyone knows these days, is that there’s an awful lot of it in every cell – meaning that the four bases (A, C, G & T) have an essentially infinite coding capacity.

That doesn’t make it any the less wonderful but it does carry a huge implication: if something you can squeeze into a single cell can carry limitless information it must be the most powerful of all storage systems.

A picture’s worth a thousand words

We looked at the storage power of DNA a few months ago (in “How Does DNA Do It?”) and noted that its storage density is 1000 times that of flash memories, that it’s fairly easy to scan text and transform the pixels into genetic code and that, as an example, someone has already put Shakespeare’s sonnets into DNA form.

Now Seth Shipman, George Church and colleagues at Harvard have taken the field several steps forward by capturing black and white images and a short movie in DNA. Moreover they’ve managed to get these ‘DNA recordings’ taken up by living cells from which they could subsequently recover the images.

Crumbs! How did they do it?

First they used essentially the text method to encode images of a human hand: assign the four bases (A, C, G & T) to four pixel colours (this gives a grayscale image: colours can be acquired by using groups of bases for each pixel). These DNA sequences were then introduced into bacteria (specifically E. coli) by electroporation (an electrical pulse briefly opens pores in the cell membrane).

The cells treat this foreign DNA as though it was from an invading virus and switch on their CRISPR system (summarized in “Re-writing the Manual of Life”). This takes short pieces of viral DNA and inserts them into the cell’s own genome in the form of ‘spacers’ (the point being that the stored sequences confer ‘adaptive immunity’: the cell has an immunological memory so it is primed to respond effectively if it’s infected again by that viral pathogen).

In this case, however, the cells have been fooled: the ‘spacers’ generated carry encoded pictures, rather than viral signatures.

Because spacers are short it’s obvious that you’ll need lots of them to carry the information in a photo. To keep track when it comes to reassembling the picture, each DNA fragment was tagged with a barcode (and fortunately we explained cellular barcoding in “A Word From The Nerds”).

Once incorporated in the bugs the information was maintained over many bacterial generations (48 in fact) and is recoverable by high-throughput sequencing and reconstruction of the patterns using the barcodes.

And the movie bit?

Simple. In principle they used the same methods to encode sequential frames.

Pictures in DNA.

Top: Using triplets of bases to encode 21 pixel colours. Images of a human hand (top) and a horse (bottom) were captured. For the movie they used freeze frames taken in 1872 by the English photographer Eadweard Muybridge. These showed that, for a fraction of a second, a galloping horse lifts all four hooves off the ground. Seemingly this won a return for the sometime California governor, Leland Stanford (he of university-founding fame) who had put a wager on geegees doing just that. From Shipman et al., 2017. You can see the movie here.

Getting the picture clear

To recap, in case you’re wondering if this is some scientific April Fools’ prank. What Church & Co. did is scan pictures and transform pixel density into the genetic code (i.e. sequences of the four bases A, C, G & T). They then made DNA carrying these sequences, persuaded bacteria to take up the DNA and incorporate it into their own genomes and, after growing many generations of the bugs, extracted their DNA, sequenced it and reconstructed the original images. By scanning sequential frames this can be extended to movies.

It’s not science fiction – but it is pretty amazing. With a droll turn of phrase Seth Shipman said “We want to turn cells into historians” and the work does have significant implications in showing something of the scope of biological memory systems.

Won’t be long before the trendy, instead of birthday presents of electronic family photo albums, are giving small tubes of bugs!

References

Shipman, S.L., Nivala, J., Macklis, J.D. & Church, G.M. (2017). CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria. Nature 547, 345–349.

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!

Reference

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

Cancer GPS?

The thing that pretty well everyone knows about cancers is that most are furtive little blighters. They kill one in three of us but usually we don’t they’re there until they are big enough to make something go wrong in the body or to show up in our seriously inadequate screening methods. In that sense they resemble heart problems of one sort or another, where often the first indication of trouble is unexpectedly finding yourself lying on the floor.

Meanwhile, out on the highways and byways you are about 75 times less likely to be killed in an accident than you are to succumb to either cancers or circulation failure. Which is a way of saying that in the UK about 2000 of us perish on the roads each year. That it’s ‘only’ 2000 is presumably because here your assailant is anything but furtive. All you’ve got to do is side-step the juggernaut and you’ll probably live to be – well, old enough to get cancer.

Did you know, by the way, that ‘juggernaut’ is said to come from the chariots of the Jagannath Temple in Puri on the east coast of India. These are vast contraptions used to carry representations of Hindu gods on annual festival days that look as though walking pace would be too much for them. So, replace the monsters on our roads with real juggernauts! Problem largely solved!!

Flagging cancer

But to get back to cancer or, more precisely, the difficulty of seeing it. After centuries of failing to make any inroads, recent dramatic advances give hope that all is about to change. These rely on the fact that tissues shed cells – and with them DNA – into the circulation. Tumours do this too – so in effect they are scattering clues to their existence into blood. By using short stretches of artificial DNA as bait, it’s possible to fish out tumour cell DNA from a few drops of 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 other cells in every cubic millimeter of blood.

There are two big attractions in this ‘microfluidics’ approach. First it’s almost ‘non-invasive’ in needing only a small blood sample and, second, it is possible that indicators may be picked up long before a tumour would otherwise show up. In effect it’s taking a biochemical magnifying glass to our body to ask if there’s anything there that wouldn’t normally be present. Detect a marker and you know there’s a tumour somewhere in the body, and if the marker changes in concentration in response to a treatment, you have a monitor for how well that treatment is doing. So far, so good.

And the problem?

These ‘liquid biopsy’ methods that use just a teaspoonful of blood have been under development for several years but there has been one big cloud hanging over them. They appear to be exquisitely sensitive in detecting the presence of a cancer – by sequencing the DNA picked up – but they have not been able to pinpoint the tissue of origin. Until now.

Step forward epigenetics

Shuli Kang and colleagues at the University of California at Los Angeles and the University of Southern California have broken this impasse by turning to epigenetics. We noted in Twenty More Winks that an epigenetic modification is any change in DNA, other than in the sequence of bases (i.e. mutation), that affects how an organism develops or functions. They’re brought about by tacking small chemical groups (commonly methyl (CH3) groups) either on to some of the bases in DNA itself or on to the proteins (histones) that act like cotton reels around which DNA wraps itself. The upshot is small changes in the structure of DNA that affect gene expression. You can think of DNA methylation as a series of flags dotted along the DNA strand, decorating it in a seemingly random pattern. It isn’t random, of course, and the target for methylation is a cytosine nucleotide (C) followed by a guanine (G) in the linear DNA sequence – called a CpG site because G and C are separated by one phosphate (p). Phosphate links nucleosides together in the backbone of DNA.

Cancer cells often display abnormal DNA methylation patterns – excess methylation (hypermethylation) in some regions, reduced methylation in others – that contributes to their peculiar behavior. It’s possible to determine the methylation profile of a DNA sample (by a method called bisulfite sequencing).

Kang & Co. developed a computer program to analyse methylation profiles from solid tumours and healthy samples in public databases and compare them to patient DNA of unknown tissue origin.

The peaks represent CpG clusters that characterize normal cells (top) and a variety of cancers. The key point is that the different patterns identify the tissue of origin (from Kang, S. et al., 2017).

The program’s called CancerLocator and in this initial study it was used to test samples from patients with lung, liver or breast cancer. In the modest words of the authors, CancerLocator ‘vastly outperforms’ previous methods – mind you, they struggle to even to distinguish most cancer samples from non-cancer samples. Nevertheless, CancerLocator’s a big step forward, not least because it can detect early stage cancers with 80% accuracy.

It’s also reasonable to expect major improvements as methylation sequencing becomes more extensive and higher resolution reveals more subtle signatures. What’s more, in principle, it should be able to detect all types of cancers – meaning that, after all so many centuries we may at last have a way of side-stepping the juggernaut.

References

Kang, S. et al. (2017). CancerLocator: non-invasive cancer diagnosis and tissue-of-origin prediction using methylation profiles of cell-free DNA. Genome Biology DOI 10.1186/s13059-017-1191-5.

The answer to … everything is …

42, as all fans of Douglas Adams and The Hitchhiker’s Guide to the Galaxy will instantly tell you. In the years before he produced his best-seller, a chance contact with Footlights had drawn me into spending many merry evenings with Douglas in The Baron of Beef public house, more or less opposite St John’s College, where he was studying – sporadically, he would doubtless have said – English.

Had a piece of work that’s just come out in The British Medical Journal been published 40-odd years earlier I suspect I would have mentioned it at one of those gatherings – early on before rational thought took alcohol-fuelled flight. It’s interesting because it says we can put off dying from the things that kill most of us (heart failure and cancer) by what Jason Gill, Carlos Celis-Morales and their pals in the University of Glasgow call ‘active commuting’. By that they mean cycling to work is good. Physical inactivity (e.g., spending happy evenings in the pub) is bad.

Had I mentioned it, rather than coming up with an entirely whimsical response to the “ultimate question of life”, Douglas would have spotted that the key to hanging on to life is “on your bike”. Just think: if Jason & Chums had got a move on, history would have been changed. Pondering all their evidence over several pints of The Baron’s best, it’s hard to imagine Douglas coming up with any title other than The Biker’s Guide to the Galaxy.

But hang on: isn’t this just another pretty useless survey?

Maybe – but for several reasons it’s hard to write it off.

First, there have been quite a few studies over the years showing that cycling is good for you.

Second, this is one was huge – so more likely to be meaningful. Using the UK Biobank data it looked for links between death and the way in which more than a quarter of a million people got to work.

Third, and the thing that really caught my eye: the key finding stuck out like the proverbial sore thumb. Usually in surveys of things that might affect our health any trends are difficult to spot: eating X makes you live 10% longer or be 5% less likely to get Y … bla, bla, bla. But here you didn’t need to peer: cycling (a ‘long distance’) to work makes you 40% less likely to die – from anything!

Below is just one bit of their data: I’ve re-drawn it with the cycling result in red but it hardly needs that to highlight the difference between it, walking (blues) and the ‘non-actives’ (green: car or public transport). It’s true, a bit of biking can help (orange: mixed mode cycling) but the really clear benefit comes from cycling (lots) – though they don’t actually say how many miles per day counts as ‘long-distance cycling.’ Modes of transport and distances were self-reported and the latter just divided into ‘long’ and ‘short’.

How you get to work impacts your life expectancy. The figure shows the risk of death from all causes as hazard ratios (ratio of the hazard rates of death): the reference (hazard ratio 1) is travel by car or public transport (green). (From Celis-Morales, C. et al., 2017).

So what of heart failure and cancer?

Perhaps not surprisingly then, commuting by cycling was also associated with a markedly lower risk both of getting heart disease or cancer and of dying therefrom. To give one specific figure: cycling to work lowers the chance of developing cancer by 45%.

It can’t be the lycra

These are horrible studies to undertake, partly because they rely on human beings telling the truth but also because of what are called ‘confounding factors.’ For example, if someone plays a lot of sport and eats sensibly, you might guess they’d be relatively healthy, regardless of how they get to work. Conversely for smoking. However, Celis-Morales & Co did their best to allow for such things and therefore to come up with results that mean something.

But, if you take their findings at face value there remains a key question that the authors do not mention: what is it about biking that’s such a life-saver (assuming you don’t get knocked-off and squashed)? It’s a real puzzle because walking is generally held to be very good for you whilst cycling is the most energy-efficient means of transport devised by man. Both activities use nearly all of your muscles, albeit that biking really works out your glutes and quadriceps, but because bikes are so efficient you use less energy.

Counting the calories

You can do the sums – i.e. work out how many calories used walking, running or cycling on Wolfram Alfra. It’s just confirmed that my daily bike commute does indeed use about half the number of calories required for the same walk.

If you take your commute as training you would suppose that expending more energy (i.e. walking rather than biking) would strengthen your heart and cardiovascular system – and indeed this study shows commuters who did more than 6 miles a week at ‘typical walking pace of three miles an hour’ slightly lowered their risk of cardiovascular disease. But cycling was far more beneficial.

As to cancer, beyond the simplistic notion that fitness = strengthening your immune system and hence capacity resist abnormal cell growth, it’s hard to see a mechanism for biking being so much better than anything else.

So, never mind the science …

Away with Ford Prefect and latter-day variants, automotive  or otherwise! On your bike!! And if you can do it with a friend on a tandem, so much the better!!! Though if you’re going to do it à deux, it might be worth recalling that the Jatravartids had the wisdom to invent the aerosol deodorant before the wheel.

Reference

Celis-Morales, C. et al. (2017). Association between active commuting and incident cardiovascular disease, cancer, and mortality: prospective cohort study. British Medical Journal 357 doi: https://doi.org/10.1136/bmj.j1456

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!

Reference

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

Now For Something Completely Different

As our faithful followers know, in these columns we adhere very strictly to science and in particular to cancer biology.

However, the time has come to reveal that we do engage in pursuits beyond science. Mainly because we need you all to support cottage industries by forking out $2.99 for our latest book Wooffie Says …

What is it? A book of short stories aimed firstly at youngsters learning to read. But the hope is that everyone will enjoy them – because they’re about cats!

But here comes the twist: each story contains a bit of science – the idea being to get kids interested as they’re enjoying the tales.

It also has a ‘good’ English’ theme.

The book is electronic so we’ve got links to make it easy to follow up each topic.

Oh, and it’s illustrated with wonderful cartoons!!

You’ll love it – and, even more important, so will all the kids in your family.

You can find it at:

https://www.smashwords.com/books/view/711108

 

 

Boldly Going

When you come across a very successful, middle-aged scientist jumping up and down shouting “This is going to be just amazing” you can only conclude that either the pressures of the life scientific have finally got to him and he’s flipped or there is something really remarkable going on. Thus my feeling this week when I noted the behaviour of Greg Hannon who now works at the Cancer Research Institute in Cambridge.

Probing further, it emerged that Hannon, who is collaborating with Xiaowei Zhuang at Harvard University in the ‘other’ Cambridge, has just been awarded a five-year grant of £20 million by the London-based charity Cancer Research UK as part of its Grand Challenge initiative – more than enough to get your jumping genes going.

But it’s the aim of the project rather than its monetary size that is truly astonishing and has almost a feel of science fiction about it. The plan is nothing less than to come up with an interactive virtual-reality map of breast cancers. That is, to reconstruct every cell that makes up a tumour, showing the different types of cell and what they are up to at any given time – meaning that the model will display the expression level of thousands of genes in each cell and the different proteins being made. Staggering.

What’s the point?

The project is driven by the fact that we have gradually come to realize that tumours are a complex mixture of cells (what’s been called the tumour microenvironment) and the signals that these cells send out and receive determine the extent of tumour growth and whether it can spread to other sites in the body (i.e. metastasize).

Where have we got to?

One approach to mapping what’s going on was laid out a couple of years ago by the converging studies of Rahul Satija and colleagues of the Broad Institute of MIT and Harvard and Kaia Achim et al. from labs in Heidelberg, Cambridge and Oxford using zebrafish embryos and worm brains, respectively.

The method has two parts:

  1. The tissue is dissociated into single cells and the power of sequencing is applied to obtain RNA sequences from each cell (revealing which genes are ‘switched on’ in that cell).
  2. The second step visualizes specific RNAs using tagged probes (fluorescently labeled RNAs that enter cells and bind to target RNAs molecules).

In essence a reference map is made by overlaying the intact tissue with a grid and matching a cell to a grid area by comparing expression of a number of ‘landmark’ genes with the fluorescence marker signal.

To do all this they devised a computational package that, using fewer than 100 landmark genes, maps hundreds of sequenced cells to their location in the tissue. In that arty way that scientists have, they named their package after Georges-Pierre Seurat, the French chappie who came up with the idea of painting in small dots of colour (though his weren’t fluorescent).

Cellular pointillism has just taken another step forward with Keren Bahar Halpern, Ido Amit and Shalev Itzkovitz at the Weizmann Institute of Science, Rehovot, Israel producing a cell-by-cell map of mouse liver, complete with RNA sequences from each cell. To be precise they mapped the hexagon-shaped units called lobules that are repeated to make up mammalian liver.

The shapes of things to come

So the next step for Hannon and his colleagues is an interactive map of a human tumour and, if you can’t wait, CLICK HERE to see their mock-up to give you some idea of what’s in store. In this synthetic video tumour cells are green, macrophages are blue and blood vessels are red.

Overwhelming?

So it’s warp factor 9 for Captain Hannon and his crew. It may be that the 3D images of tumours will look a bit the virtual graphics that the astrophysicists fob off on us whilst pretending they have some idea what a star’s doing umpti-zillion light years away. But in fact, rather than boldly going where no man has gone before“, this cellular journey is better summed up by Marcel Proust The real voyage of discovery consists not in seeking new landscapes, but in having new eyes” – the new eyes being the stunning combination of methods that permits 3D interrogation of individual cells.

Will this phase of the Grand Challenge produce overwhelming amounts of data? Undoubtedly. But, if we are to understand how living things work we have to front up to the complexity of nature. We then have to hope we are smart enough to resolve the crucial from the detail.

References

Satija, R. et al. (2015). Spatial reconstruction of single-cell gene expression data. Nature Biotechnology 33, 495–502.

Achim, K. et al. (2015). High-throughput spatial mapping of single-cell RNA-seq data to tissue of origin. Nature Biotechnology 33, 503–509.

Halpern, K. B. et al. (2017). Nature 542, 352–356.

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.

raleighs_first_pipe_in_england-jpeg

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!

References

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.