Bigger is Better

“Nonsense!” most males would cry, quite logically, given that we spend much of our time trying to persuade the opposite sex that size doesn’t matter. But we want to have it both ways: in the macho world of rugby one of the oldest adages is that ‘a good big ’un will always beat a good little ’un’.  Beethoven doubtless had a view about size – albeit unrecorded by history – but after he’d written his Eroica symphony, perhaps the greatest revolutionary musical composition of all, his next offering in the genre was the magical Fourth – scored for the smallest orchestra used in any of his symphonies. And on the theme of small can be good, the British Medical Journal, no less, has just told us that if we cut the size of food portions and put ’em on smaller plates we’ll eat less and not get fat!

Is bigger better?

Is bigger better?

All of which suggests that whether bigger is better depends on what you have in mind. Needless to say, in these pages what we have in mind is ‘Does it apply to cancer?’ – that is, because cancers arise from the accumulation in cells of DNA damage (mutations), it would seem obvious that the bigger an animal (i.e. the more cells it has) and the longer it lives the more likely it will be to get cancer.

Obvious but, this being cancer, also wrong.

Peto’s Paradox

The first person to put his finger on this point was Sir Richard Peto, most famous for his work with Sir Richard Doll on cancer epidemiology. It was Doll, together with Austin Bradford Hill, who produced statistical proof (in the British Doctors’ Study published in 1956) that tobacco smoking increased the risk of lung cancer. Peto joined forces with Doll in 1971 and they went on to show that tobacco, infections and diet between them cause three quarters of all cancers.

Whenever this topic comes up I’m tempted to give a plug to the unfortunate Fritz Lickint – long forgotten German physician – who was actually the first to publish evidence that linked smoking and lung cancer and who coined the term ‘passive smoking’ – all some 30 years before the Doll study. Lickint’s findings were avidly taken up by the Nazi party as they promoted Draconian anti-smoking measures – presumably driven by the fact that their leader, Gröfaz (to use the derogatory acronym by which he became known in Germany as the war progressed – from Größter Feldherr aller ZeitenGreatest Field Commander of all Time) was a confirmed non-smoker. Despite his usefulness, Lickint’s political views didn’t fit the ideology of the times. He lost his job, was conscripted, survived the war as a medical orderly and only then was able to resume his life as a doctor – albeit never receiving the credit he deserved.

Returning to Richard Peto, it was he who in 1975 pointed out that across different species the incidence of cancer doesn’t appear to be linked to the number of cells in animal – i.e. its size.   He based his notion on the comparison of mice with men – we have about 1000 times the number of cells in a mouse and typically live 30 times as long. So we should be about a million times more likely to get cancer – but in fact cancer incidence is another of those things where we’re pretty similar to our little furry friends. That’s Peto’s Paradox.

It doesn’t seem to apply within members of the same species, a number of surveys having shown that cancer incidence increases with height both for men and women. The Women’s Health Initiative found that a four inch increase in height raised overall cancer risk by 13% although for some forms (kidney, rectum, thyroid and blood) the risk went up by about 25%. A later study found a similar association for ovarian cancer: women who are 5ft 6in tall have a 23% greater risk than those who only make it to 5 feet. A similar risk links ovarian cancer to obesity (i.e. a rise in body mass index from 20 (slim) to 30 (slightly overweight) puts the risk up by 23%). Statistically sound though these results appear to be, it’s worth nothing that, as my colleague Paul Pharoah has pointed out, these risk changes are small. For example, the ovarian cancer finding translates to a lifetime risk of about 16-in-a-1000 for shorter women going up to 20-in-a-1000 as they rise by 6 inches.

It’s true that there may be a contribution from larger animals having bigger cells (whale red blood cells are about twice as big as those of the mouse) that divide more slowly but at most that effect seems small and doesn’t fully account for the fact that across species the association of size and age with cancer breaks down: Peto’s Paradox rules – humans are much more likely to get cancer than whales.

What did we know?

Well, since Peto picked up the problem, almost nothing about underlying causes. The ‘almost’ has been confined to the very small end of the scale and we’ve already met the star of the show – the naked mole rat – a rather shy chap with a very long lifespan (up to 30 years) but who never seems to get cancer. In that piece we described the glimmerings of an explanation but, thanks to Xiao Tian and colleagues of the University of Rochester, New York we now know that these bald burrowers make an extraordinarily large version of a polysaccharide (a polymer of sugars). These long strings of glucose-like molecules (called hyaluronan) form part of the extracellular matrix and regulate cell proliferation and migration. They’re enormous molecules with tens of thousands of sugars linked together but the naked mole rat makes versions about four times larger than those of mice or humans – and it seems that these extra-large sugar strings restrict cell behaviour and block the development of tumours.

Going up!

Our ignorance has just been further lifted with two heavyweight studies, one from Lisa Abegglen, Joshua Schiffman and chums from the University of Utah School of Medicine who went to the zoo (San Diego Zoo, in fact) and looked at 36 different mammalian species, ranging in size from the striped grass mouse (weighing in at 50 grams) to the elephant – at 4,800 kilogram nearly 100,000 times larger. They found no relationship between body size and cancer incidence, a result that conforms to Peto’s paradox. Comparing cancer mortality rates it transpires that the figure for elephants is less than 5% compared with the human range of 11% to 25%.

107 final pic

Cancer incidence across species by body size and lifespan. A selection of 20 of the 36 species studied is shown. Sizes range from the striped grass mouse to the elephant. As the risk of cancer depends on both the number of cells in the body and the number of years over which those cells can accumulate mutations, cancer incidence is plotted as a function of size (i.e. mass in grams × life span, years: y axis: log scale). Each species is represented by at least 10 animals (from Abegglen et al., 2015).

It can be seen at a glance that cancer incidence is not associated with mass and life span.

The Tasmanian devil stands out as a remarkable example of susceptibility to cancer through its transmission by biting and licking.

How does Jumbo do it?

In a different approach to Peto’s Paradox, Michael Sulak, Vincent Lynch and colleagues at the University of Chicago looked mainly at elephants – more specifically they used DNA sequencing to get at how the largest extant land mammal manages to be super-resistant to cancer. In particular they focused on the tumor suppressor gene P53 (aka TP53) because its expression is exquisitely sensitive to DNA damage and when it’s switched on the actions of the P53 protein buy time for the cell to repair the damage or, failing that, bring about the death of the cell. That’s as good an anti-cancer defence as you can imagine – hence P53’s appellation as the ‘guardian of the genome’. It turned out that elephants have no fewer than 20 copies of P53 in their genome, whereas humans and other mammals have only one (i.e. one copy per set of (23) chromosomes). DNA from frozen mammoths had 14 copies of P53 but manatees and the small furry hyraxes, the elephant’s closest living relatives, like humans have only one.

The Utah group confirmed that elephants have, in addition to one normal P53 gene, 19 extra P53 genes (they’re actually retrogenes – one type of the pseudogenes that we met in the preceding post) that have been acquired as the animals have expanded in size during evolution. Several of these extra versions of P53 were shown to be switched on (transcribed) and translated into proteins.

Consistent with their extra P53 fire-power, elephant cells committed P53-dependent suicide (programmed cell death, aka apoptosis) more frequently than human cells when exposed to DNA-damaging radiation. This suggests that elephant cells are rather better than human cells when it comes to killing themselves to avoid the risk of uncontrolled growth arising from defective DNA.

More genes anyone?

Those keen on jumping on technological bandwagons may wish to sign up for an extra P53 gene or two, courtesy of genetic engineering, so that bingo! – they’ll be free of cancers. Aside from the elephant, they may be encouraged by ‘super P53’ mice that were genetically altered to express one extra version of P53 that indeed significantly protected from cancer when compared with normal mice – and did so without any evident ill-effects.

We do not wish to dampen your enthusiasm but would be in dereliction of our duty is we did not add a serious health warning. We now know a lot about P53 – for example, that the P53 gene encodes at least 15 different proteins (isoforms), some of which do indeed protect against cancer – but there are some that appear to act as tumour promoters. In other words we know enough about P53 to realize that we simply haven’t a clue. So we really would be playing with fire if we started tinkering with our P53 gene complement – and to emphasise practicalities, as Mel Greaves has put it, we just don’t know how well the elephants’ defences would stack up if they smoked.

Nevertheless, on the bright side, light is at long last beginning to be shed on Peto’s Paradox and who knows where that will eventually lead us. Meanwhile Richard Peto’s activities have evolved in a different direction and he now helps to run a Thai restaurant in Oxford, a cuisine known for small things that pack a prodigious punch. Bit like Beethoven’s Fourth you could say.

a-gem-of-a-find-in-oxford

References

Peto, R. et al. (1975). Cancer and ageing in mice and men. British Journal of Cancer 32, 411-426.

Doll, R. and Peto, R. (1976). Mortality in relation to smoking: 20 years’ observations on male British doctors. Br Med J. 2(6051):1525–36.

Maciak, S. and Michalak, P. (2015). “Cell size and cancer: A new solution to Peto’s paradox?”. Evolutionary Applications 8: 2.

Doll, R. and Hill, A.B. (1954). “The mortality of doctors in relation to their smoking habits”. BMJ 328 (7455): 1529.

Doll, R. and Hill, A.B. (November 1956). “Lung cancer and other causes of death in relation to smoking; a second report on the mortality of British doctors”. British Medical Journal 2 (5001): 1071–1081.

Tian, X. et al. (2013). High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat. Nature 499, 346-349.

Abegglen, L.M., Schiffman, J.D. et al. (2015). Potential Mechanisms for Cancer Resistance in Elephants and Comparative Cellular Response to DNA Damage in Humans. JAMA. doi:10.1001/jama.2015.13134.

Sulak, M., Lindsey Fong, Katelyn Mika, Sravanthi Chigurupati, Lisa Yon, Nigel P. Mongan, Richard D. Emes, Vincent J. Lynch, V.J. (2015). TP53 copy number expansion correlates with the evolution of increased body size and an enhanced DNA damage response in elephants. doi: http://dx.doi.org/10.1101/028522.

García-Cao, I. et al. (2002). ‘Super p53’ mice exhibit enhanced DNA damage response, are tumor resistant and age normally. EMBO Journal 21, 6225–6235.

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Guess Who’s Coming to Dinner?

 

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

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

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

Player’s biography

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

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

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

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

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

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

So not all pseudogenes are extinct then?

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

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

106 pic

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

Interfering RNA

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

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

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

First but not last

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

References

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