p53 (21 page)

Glantz’s paper is littered with the names of scientists
paid by Big Tobacco to carry out research on its behalf, frequently with the explicit aim of discrediting
the evidence of a causal link between smoking and cancer, or to write letters to medical journals or popular newspapers for the same purpose. Sifting through the wealth of information the tobacco
industry has been obliged to publish, Glantz found evidence that by the early 1990s, BAT had identified p53 as being especially important, with ‘more papers published on it than any other
topic on cancer’. The company monitored research on the gene and pressed its paid scientists for intelligence about what their colleagues were discovering and for advance copies, if possible,
of relevant papers submitted for publication. Furthermore, BAT regarded information about the research organisations it supported as confidential and advised individual scientists that they were
‘free to publish their work without further reference’ to the company. BAT and others also appear to have anticipated Pfeifer and Denissenko’s findings about the effects of
tobacco tar on p53, and to have worked on a strategy to discredit them in advance.

The idea to challenge the scientists’ interpretation of data from the IARC database seems to have come from Jim Parry, who published the two critical papers in
Mutagenesis.
But
though Hainaut was relieved when he first discovered the evidence of skulduggery, he found himself unable to dismiss the attacks out of hand. ‘I think, to be fair, the tobacco industry
criticism had a point,’ he told me. ‘Our data were based on putting together bits and pieces of our database from studies which were not aimed at demonstrating what we wanted to
demonstrate. We had data on never-smokers, for example, that were scattered around something like 20 different papers, none of which had the scope on its own to demonstrate what we wanted to show;
it was just by putting them together that they made the point.’

He and his fellow researchers decided to go back to the drawing board and come up with an analysis that was
rigorous and powerful enough to prove their case. ‘It
took us two years, but we did it and we published the paper in 2005 in
Cancer Research
,’ said Hainaut. ‘Nobody now can say the connection is not there; it’s really
watertight. I think the whole story is now behind us, but that’s what it took! And maybe in that respect it was a good thing we had this attack, because otherwise we might not have done the
paper.’

Finally nailing Big Tobacco more than half a century after the first warnings of the dangers of smoking was a triumph for p53 and public health, but it’s not the only one. The mutant p53
database is proving a rich resource for the disease detectives, who are finding a growing list of carcinogens that leave their unique fingerprints on the cancers they cause. Besides tobacco, mouldy
peanuts on liver cancer and sunlight on skin are just two of the direct relationships the scientists have been able to work out in forensic detail.

***

Liver cancer is the seventh most common cancer worldwide, but in South East Asia and sub-Saharan Africa, where the great majority of cases occur, it kills more people every
year than any other tumour type. In these regions Hepatitis B, which is a major risk factor for liver cancer everywhere, is extremely widespread – passed between sexually active adults and
from mother to child, much like HIV. And like the AIDS virus, too, it can wreak havoc in a person’s body without them being aware of the infection, and become endemic in communities. Hep B
generally takes many years to cause liver cancer, but in the high-incidence countries of Asia and Africa people’s risk of getting the disease is compounded by exposure also to aflatoxin, a
poison produced by the fungus
Aspergillus
that flourishes on peanuts and grains stored in warm, damp conditions without adequate ventilation.

Aflatoxin is a known carcinogen and was one of the chemicals investigated by Curt Harris’s lab in the late 1980s and early ’90s for its mechanism of action in human cells. As with
BaP in tobacco tar, Harris knew that aflatoxin is metabolised and transformed in cells into a substance that sticks to DNA and causes mutations. But it was work he did
with
colleagues in China, analysing the genetic mutations in liver tumours in Qidong county, on the north side of the Yangtze River opposite Shanghai, that showed the poison at work in the real world
and pointed the finger at p53 as being the target for mutation. Rates of liver cancer in the county were exceptionally high; so too was people’s exposure to aflatoxin from mouldy grains and
beans in their diet, and the researchers were struck by the frequency of an unusual mutation in p53 at codon 249. This resulted in the building blocks of the gene being swapped, a G to a T –
the same as with tobacco tar, but in a different hot spot on the gene. Could this be the fingerprint of aflatoxin?

As the paper describing Harris and his colleagues’ findings and suggesting such a possibility was about to go to press, a visiting scientist to Harris’s lab at the National Cancer
Institute mentioned casually that another group, working in South Africa, had also discovered an unusual p53 mutation in liver tumours, but were unsure of its significance or what to do with their
findings. Realising that this strengthened their case for a direct link between aflatoxin and p53 in liver cancer, Harris pushed the other group, led by Mehmet Ozturk, to write up their research in
double-quick time so that the two papers could be published together, and they came out back to back in
Nature
in April 1991.

The coincidence of aflatoxin exposure and a distinctive p53 mutation in liver-cancer patients soon became apparent in many other warm, humid places with poor storage for crops. But what was
going on in the machinery of their cells? Pierre Hainaut joined the quest to find out. Despite his initial unease at being drawn into the tobacco and cancer controversy, Hainaut is a natural-born
sleuth, never happier than when he is doing research on the front line, where faulty tumour-suppressor genes are affecting the lives of real people. He has followed the fingerprints of p53 from
China to Brazil, Iran to West Africa and South East Asia, and many other countries. With aflatoxin, his research has
focused mainly on Mali, The Gambia and Thailand –
three countries where liver cancer is a huge problem. Over the years he and others working on this issue, including Harris and Pfeifer, have revealed a devilish relationship between aflatoxin, the
ubiquitous Hep B virus and p53, as they co-operate to cause liver cancer.

First the scientists worked out how Hep B virus on its own can lead to liver cancer. A viral gene – known simply as ‘x’ because for a long time no one had a clue what it did
– codes for a protein that has a dual function: one end of the protein encourages proliferation of the liver cells it’s infecting; the other end promotes apoptosis, cell death. In this
way the virus tries to maintain some kind of balance in the population of infected liver cells. But in so doing, it causes cycles of inflammation, damage and repair to the liver that result in
cirrhosis – a liver greatly enlarged and distorted by scar tissue and lumps, or nodules, of regenerated cells.

‘These cycles of destruction and regeneration can go on for a while, and sometimes they can kill people – they can die from cirrhosis of the liver without getting cancer,’
explained Hainaut. ‘But at some point, in the absence of mutant p53, what happens to people with chronic liver disease and cirrhosis is that HBx becomes accidentally integrated into the
genome of the liver cells. At that point it loses it pro-apoptotic part, and what remains is just the part that activates proliferation: the cells then escape destruction and are on their way to
cancer. This is why cancer develops as a sequel to cirrhosis in the context of wild-type p53.’

Harris’s group found also that HBx protein sticks to p53 protein, forming a complex in much the same way as SV40 does with p53. They assumed that in so doing HBx had a similar effect of
crippling the tumour-suppressor function of p53, and that this was one of the driving forces towards cancer. However, very recent research by Hainaut and his colleagues in West Africa suggests this
assumption is wrong; it has the relationship between the two proteins back to front,
for what really seems to be happening is that p53 is blocking the ability of the virus
protein, HBx, to trigger apoptosis, while leaving its ability to drive proliferation of cells intact. The crucial point here is that, in real life, the bond between p53 and HBx that transforms the
viral protein is only really strong when p53 has the aflatoxin-induced mutation, at codon 249. Then the brakes are off and the liver is especially vulnerable to cancer. ‘The risk of having
liver cancer for someone who is a chronic carrier of Hep B is about 5-7 times compared to a non-chronic carrier,’ Hainaut told me. ‘The risk of getting liver cancer with just aflatoxin
is very difficult to measure, but is probably no more than twofold. However, the risk of having liver cancer if you have the two is at least 20 times, and some measures suggest it is up to 60 times
greater than usual. So it’s truly multiplicative.’

The revelation that mutant p53 transforms the function of the virus rather than the other way round has also helped to explain an abiding mystery in African liver-cancer patients. When someone
infected with Hep B virus finally succumbs to liver cancer, he or she generally has signs of advanced cirrhosis from years of damage and repair. ‘This is the rule in the Western world,’
said Hainaut. ‘The patient who doesn’t develop cirrhosis before liver cancer is really the exception.’ But this is not what they have found among patients with liver cancer in
Africa, despite chronic infection with Hep B. ‘I would say that maybe 15 per cent have cirrhosis beforehand, and then a number of them develop cirrhosis during the proliferation of cancer, as
a sort of secondary response of the liver to the inflammatory state, but it does not precede cancer.’

Hainaut’s theory is that by blocking the virus’s killer function, the mutant p53 prevents the regular cycles of inflammation, damage and repair that cause the scars and nodules of
cirrhosis. Thus, paradoxically, aflatoxin exposure can be protective of people with chronic Hep B infection, often for years, until other events in the ordinary course of
living render them vulnerable to cancer. ‘We could never understand why we have so little liver cirrhosis in these populations. It’s something I observed about 15 years
ago – we had very few patients with cirrhosis. The common response was, “Ah, you’re not looking for them . . . They’re not reporting to doctors, so detection is not good . .
. The diagnosis is not accurate,” and so on. But since then we’ve done a few cohort studies (which follow a group of people who share common characteristics and lifestyles) and still we
find most patients presenting with liver cancer without any trace of cirrhosis beforehand.’

In their studies among liver-cancer patients in Thailand, Hainaut and his team found the same phenomenon – those who had the aflatoxin mutation as well as Hep B infection had no signs of
cirrhosis. But though aflatoxin-mutated p53 may be protective of livers in the short term, the case for controlling the offending fungus to reduce the burden of liver cancer is overwhelming, as
events in Mali have shown serendipitously. Poring over the cancer register in the capital city, Bamako, very recently, Hainaut and colleagues faced another mystery – liver-cancer cases seemed
to be plummeting. In the 15 years since the late 1990s, the rate of new cases had declined by about 75 per cent. They looked for flaws and biases in the records, but could find nothing obvious to
explain away the dramatic figures.

On further investigation they discovered that in the mid-1990s, the agriculture ministry had started a programme to prevent aflatoxin contamination of the country’s crops. The primary
motivation was not public health but economics: Mali wanted to export its crops for animal feed and needed to comply with international regulations. But this has had far-reaching consequences for
the man and woman in the street, said Hainaut. ‘The first thing is that the contamination of food has decreased; and second, most of the crop production has been diverted towards export, so
the diet has changed.’ Today, the exposure to aflatoxin
of people in Mali is only a tiny fraction of the exposure of people in The Gambia, where rates of liver cancer
remain as high as ever.

But there is a downside to this story: as Hainaut and his colleagues predicted, doctors are beginning to see more people with liver cirrhosis in Mali than ever before, as Hep B is still
widespread but the factor that keeps the offending viral gene under control – aflatoxin-mutated p53 – is no longer so common.

The great appeal of molecular epidemiology to those involved is that it is often swiftly and directly applicable to real life, and this is the case with liver cancer and mouldy grains. Having
worked out the relationship between aflatoxin, p53 and Hep B virus, the scientists find they can read much of what is going on in a person’s liver with a blood test. ‘The point is that
when material is being cleared from the liver it goes either into the bile or the blood. It can’t go anywhere else – there’s no direct route to the outside world like in the
digestive tract or the lungs,’ explained Hainaut. ‘That means that every bit of DNA from liver cells ends up in the bloodstream. And the liver is such a massive organ that a large part
of the free-circulating DNA that you find in the blood comes from the liver.’