p53 (28 page)

However, both Lozano’s and Jacks’ mice showed that indeed the mutant protein does pool in real life. They furnished compelling evidence, too, that gain of function – by some
p53 mutants at least – is a real phenomenon and not an artefact produced by force. The two groups used different strains of mice in their experiments, which meant that the mutant genes were
operating against a variety of background environments, thus adding weight to their findings. As controls for their experiments, both groups used a mouse with one wild-type allele and the other p53
allele missing altogether; these control mice, too, were prone to cancer, but mutation would be no part of the picture, allowing the researchers to see, by comparison with their LFS-like mice, what
effect, if any, mutation had on the types of tumours that developed.

So what did they find? As one would expect with living creatures, the different mice met different fates. They all – those with one mutant p53 allele (the LFS-like mice) and those with one
missing allele (the controls) – developed tumours. In all Jacks’ mice with mutant p53 the range of tumours that developed was different from that seen in the control mice. In
Lozano’s R175H mutants, on the other hand, the tumours that developed were similar to those of their controls, but they were much more aggressive: they spread readily to the lymph nodes,
lungs, liver and brains of the mice, while the tumours of the controls did not metastasise.

‘To me that was the most convincing experiment,’ Lozano commented. ‘When you compare those two mice and one has a tendency to produce tumours that metastasise and the other one
doesn’t, how can you argue against gain of function? I mean, you can’t!’ What finally clinched the argument for most of the p53 community – including some die-hard sceptics
of gain of function – was that both Lozano’s and Jacks’ mice developed some novel tumours that are never seen in knock-out mice with
no
p53. This could mean only one
thing: that the mutant was doing more than simply hobble the wild-type allele and shut down its protective functions – clearly it had a life of its own.

These and other mouse models have allowed researchers gradually to build a picture of how the mutants work and how they interact with wild-type p53. Context, it seems, is all-important. Not only
do the mutants differ from one another in their actions, but they behave differently in one cell type, tissue or organ from another, and in one strain of mouse (and presumably one human being) from
another. Timing, too, is critical: in some cancers p53 mutation is an early event; in others it occurs when the tumour is already advanced, and may (as in the case of colon cancer) mark the turning
point between a benign growth and malignancy.

As for their mechanism of action, it seems that p53 mutants sometimes co-operate with other oncogenes, such as Ras, to drive the growth of tumours. Sometimes they achieve the same effect through
interaction with another protein in the cell – notably one or other of p53’s closest relatives, p63 or p73, which share some of its tumour-suppressor characteristics and can be hobbled
by the mutant. Some mutants can, like wild-type p53, switch on and orchestrate the activity of other genes. However, this is a travesty of healthy behaviour: the genes switched on by mutant p53 are
not the same genes that are controlled by the wild-type tumour suppressor, and can have the opposite effect. And a distinctive characteristic of many of the mutants is that they make cells
extremely resistant to self-destruct signals. This not only encourages the growth of tumours, but makes them very difficult to treat, since most anti-cancer therapies are designed to trigger the
apoptosis response.

An unexpected finding, made by Gigi Lozano and her group, is that the over-expression of mutant p53 protein is not an intrinsic property of the mutant gene, as had been assumed. The pooling of
the protein occurs only in tumour cells, while in the normal cells surrounding the tumour and beyond, the protein is at barely detectable levels. This implies that, like the wild-type protein, the
mutant is being regularly produced and degraded in the normal course of events until something occurs to release it from the loop. Though theories abound, no one yet knows why or how this happens
– only that the mutant protein
has
to be over-expressed to be able to act as a growth promoter.

VINDICATION

As Varda Rotter’s steadfast insistence on the importance of mutant p53 has been vindicated and the spotlight has swung back in this direction, enormous effort is being
made to understand its biology. ‘Over the last five years alone,’ wrote Carol Prives and William Freed-Pastor in a review for Cold Spring Harbor Laboratory Press in 2012, ‘p53
mutants have been found to actively contribute to tumor proliferation, survival, limitless replication, somatic cell reprogramming (i.e. turning differentiated body cells back towards stem cells),
genomic instability, inflammation, disruption of tissue architecture, migration, invasion, angiogenesis (development of a blood supply to a tumour), and metastasis.’ They concluded that this
confirms mutant p53’s central role in the development of malignant tumours, with an impact on nearly all of the ‘hallmarks of cancer’– the list of 10 defining
characteristics of all cancers – proposed by Bob Weinberg and Doug Hanahan in 2000.

It also makes the aberrant protein a prime target for therapy, as scientists and Big Pharma alike look for new, more effective ways to treat people with cancer that do not do such devastating
damage to the body’s normal, fast-dividing cells at the same time.

***

As a mouse man, Larry Donehower of Baylor College of Medicine in Houston is used to dropping bombshells. Working closely with his colleague Allan Bradley, he was, in 1992, the
first person to create a p53 knock-out mouse. Using the technique recently developed by Capecchi, Evans and Smithies, he was able to delete the gene from mouse embryonic stem cells and then implant
the developing embryos successfully into the womb of a female mouse for gestation. When he turned up to present his findings at a p53 meeting that year, the excitement was palpable and most people
had a pretty good idea of what he would say. After all, it was not long since the normal gene had been revealed as a tumour suppressor, not an oncogene; it had been found in almost every
multi-cellular organism, conserved in evolution, unchanged in form and function, since the dawn of time; and it had recently been dubbed ‘guardian of the genome’. Surely
Donehower’s mice would show that without it, life was not sustainable?

But this was not what he had come to say. His genetically engineered animals were fine. Not only had they survived without the protection of the guardian the period of explosive growth, cell
division and differentiation that turns an embryo into a pup, but they had no signs of physical deformities or cancerous growth. Donehower’s audience was stunned.

David Lane remembered the occasion vividly when I interviewed him for this book on the fringes of a big conference in Liverpool. ‘We were all in a very triumphant mood as a community.
“p53 is now the most important bloody protein in the world and all you guys can get stuffed!”, you know? “Who’s been telling us that we’ve been wasting our time for
the last 10 years?” It felt exciting and good,’ he recalled with a grin. ‘We were having this big p53 workshop in the US, and instead of just 20 people coming,
200
were
coming. There were lots of positive data . . . loads of people finding mutations, and everything was now very convincing. Then Larry stands up and says, “I’ve made a knock-out mouse and
there are some interesting things about this mouse . . . First of all it’s completely viable. There are no defects that I can see – and it certainly hasn’t got any cancer!”
Everybody went, “Uh oh!”’ Lane leaned back in his chair and gave a huge laugh of incredulity. ‘Of course, I guess it was about two months after the conference, Larry started
to see massive development of tumours in all the animals and there was a collective sigh of relief . . . Poor mice, but lucky us!’ Lane paused to reflect over the 20 years since that meeting.
‘It was incredible, actually. And, of course, having the knock-out as a tool has made an enormous difference to everything.’

Donehower had been as stunned as everyone else by his initial results, which had ramifications beyond the purely scientific. The colossal effort that goes into such research and the money poured
in to support it generate high expectations and heavy pressure for exciting results. When ‘nothing’ happens, panic sets in and doubt ripples across the whole community. In time,
however, all Donehower’s knock-out mice did indeed succumb to cancer, surviving for less than five months compared with around 30 months for normal mice of the same genetic background.
Further research also revealed that p53 knock-out mice have much smaller litters than normal, suggesting the gene plays a part somewhere in reproduction. And it has flagged up a vital role for p53
in regulating metabolism, which is one of the hottest topics of investigation at the moment.

But back to the story of ageing. In 2002, Larry Donehower and his team dropped their second bombshell when they made a mistake with one of their experiments and got a mighty surprise. They were
trying to make a knock-out mouse using a different technique from before, but they ended up instead with a mouse in which the still-present p53 gene was hyperactive. Sure enough, the creatures
proved well protected from cancer, as the researchers would have predicted. What none of them expected to see, however, was that they aged exceptionally fast. In just a few months, they looked like
very old mice. ‘They had hunchback spines, ruffled fur, grey hair; things like that. And they lived only about two-thirds of their normal life span,’ Donehower told me when I spoke to
him at a p53 meeting in New York. ‘Some of the most interesting findings in science are accidental, actually. They’re not what you’re looking for or expecting, and this was very
surprising.
Nature
published it in 2002. Now this accidental finding is opening up a whole new area of research about how this very important cancer gene can also modify the ageing
process.’

People have known for a long time that ageing and cancer are related, in that the chances of getting cancer increase with age. But not even the scientists suspected they might be two sides of
the same coin, sharing a common mechanism in which the scales can be tipped either way. In other words, that wrinkled skin, thinning bones and failing organs may be the price we pay in the long run
for holding cancer at bay. Donehower’s findings, however, left room for a smidgen of doubt about the role of p53, since the ‘accident’ that produced the hyperactive version also
knocked out a stretch of DNA upstream of the tumour-suppressor gene. The possibility could not be ruled out that something here might be responsible for the premature ageing. But soon another lab,
run by Heidi Scrable of the University of Virginia at Charlottesville, provided new evidence that Donehower’s original hunch was right. She and her team created a mouse model in which the
only change to its DNA was the replacement of one allele of p53 with a naturally occurring hyperactive version of the gene, and found the same thing – premature ageing and death.

Donehower, Scrable and others working in this compulsively intriguing field have gradually pieced together the picture of how this can happen. A hormone known as insulin-like growth factor 1
(most often represented as IGF-1) that, not surprisingly, plays a central role in the growth and proliferation of cells, has long been known to promote ageing too in all manner of organisms, from
fruit flies and nematode worms to mice. By tinkering with the strength of the signals this hormone sends out to the cells, researchers have managed to manipulate the life span of these creatures.
The effect is most obvious in flies and worms, which live considerably longer when IGF-1 signalling is dampened down and shorter when it is amplified.

Scrable and her team found that the hyperactive p53 in their engineered mice stimulated hyperactivity of the growth hormone too. The amplified signals from IGF-1 in turn triggered the mechanism
designed to bring runaway cells under control by driving them into senescence, or irreversible arrest. This, of course, is tumour suppression at work, and is orchestrated by ‘regular’
p53. To that extent it was an appropriate, and clearly beneficial, response. But senescent cells can become dysfunctional and as they accumulate in the tissues they begin to cause trouble of their
own.

Unlike cells that have been driven to suicide by apoptosis, senescent cells remain alive and active – and, significantly, they alter the micro-environment of the tissues by secreting
proteins that communicate with neighbouring cells and even with distant organs. Some of these proteins are important for tumour suppression – for example, they inhibit the development of new
blood vessels which might feed a developing tumour. But as they metabolise in the normal course of events, senescent cells also produce large amounts of material that seeps into the surrounding
tissue. ‘This begins to chew up the extra-cellular matrix – you know, the stuff that keeps cells glued together,’ said Judith Campisi, who studies senescence at the Buck Institute
for Research on Aging in Berkeley, California, when I spoke to her at the same New York meeting as Larry Donehower. ‘The major extra-cellular molecule that keeps your skin looking young is
collagen. And sure enough, senescent cells produce molecules that destroy collagen.’ Hence wrinkles.