Looking for clues

As part of Cancer Grand Challenges team Mutographs, Balmain helped find (or rediscover) the answer. He was one of the few people it didn’t surprise.

As the name suggests, Mutographs was all about mutations. In 2017, we funded the team through Cancer Grand Challenges to combine decades of advances in understanding mutations into a new approach for preventing cancer. Rather than testing potential risk factors to see if they could cause mutations, the idea was to look for patterns of mutations (mutational signatures) that might help us uncover risk factors we can’t see.

There was lots of evidence to recommend that approach, although no one had tried it before. Oesophageal cancer, for example, is far more common in some countries than in others, which suggests that there must be local culprits to blame.

In essence, Mutographs approached cancer like detectives at a crime scene. Led by the Wellcome Sanger Institute’s Professor Sir Mike Stratton, the team dusted thousands of cancer cells for the fingerprints that would point to the exposures that damaged them.

“It’s the forensics of cancer,” explains Balmain. “When something in the environment mutates DNA, it causes a certain kind of pattern of mutations. You can sequence tumours to distinguish mutations caused by sunlight, or that have a contribution from sunlight, from mutations that have a contribution from smoking.”

To find new mutational signatures linked to cancer, a team led by Stratton, Paul Brennan and Ludmil Alexandrov embarked on a huge epidemiological study. They collected samples from thousands of patients across eight cancer types and five continents. That work identified important new mutational signatures linked to kidney cancer, but it also showed something strange  – extreme variations in rates of oesophageal cancer weren’t linked to any mutational signatures at all.

That meant there had to be something else driving oesophageal cancer that wasn’t directly altering DNA.

To delve into this further, Balmain’s team turned to the lab. They set out to identify the exact mutational signatures of 20 potentially cancer‑causing chemicals by recording what they did to mouse DNA.

Again, the team noticed something strange: 17 of the 20 chemicals didn't leave any signature, any fingerprints, at all.

“They caused cancer,” says Balmain, “but they didn't cause mutations.”

The tumours caused by these chemicals didn’t look any different from the cancers some mice develop in old age – they just appeared much earlier. Something else was happening, and it was ‘waking up’ inactive cells that had already mutated in the normal course of a mouse’s life.

That reminded Balmain of an idea that had been dormant for decades. He first specialised in cancer research in the 1970s, before it was possible to track mutations the way we do today. Back then, as they had for decades before, scientists focused more on what on what they called cancer promoters, or other factors needed to turn a cell with the potential to become cancerous into a cancer cell.

Like a mutation in your DNA, that idea had never gone away – to someone like Balmain, who knew how to look for it, it was everywhere. But it needed something else to fully activate it. That’s exactly what Mutographs had done.

Shifting focus

Balmain knew it was time to change tactics.

“Mutations are essential, absolutely essential for cancer to start,” explains Balmain. “But in many cases, they’re not going to do any harm without any promoters. So we really need to understand the promoters.”

That’s what Balmain is now doing as one of the co-leads of Cancer Grand Challenges Team PROMINENT – using the very same tools that shifted the focus from promotion to mutation to investigate them both together.

“We can use our genetic tools like a microscope and see mutated cells expanding and almost breathing in response to different promoters,” he says. “Some promoters are like a sharp knife - they pinpoint a target, a certain pathway, and they cause the cells to grow. Other promoters are like hitting cells with a sledgehammer – they create damage that needs repairing. Wounding can be a very efficient promoter.”

We can see that happening in lung cancers caused by smoking, which causes some of the clearest mutations of all. Balmain calls tobacco smoke “the perfect carcinogen”, because it directly damages DNA (leaving a mutational signature called SBS4) and it leads to cancer-promoting changes around the cells it mutates. 

Those ‘promotive’ effects come from particles in tobacco smoke that cause inflammation in the lining of the lungs, a wound healing response that can disrupt the immune system over time. Without that, cigarettes would be less likely to cause cancer. 

That's a vital point: the mutations caused by smoking could remain in someone’s lungs for their whole lives. But stopping smoking gives the “wounds” surrounding those cells a chance to heal.

Promotion and the immune system

That makes Professor Yi Feng’s research at the University of Edinburgh particularly important. Before she turned her attention to cancer, she studied how the body repairs wounds.

“We started from the fact that the same immune cells seem to play different roles in wound healing and the initial stages of cancer development,” she explains. 

Feng’s lab studies skin cells using 0 to 5-day-old zebrafish larvae, which, on the one hand, are genetically similar to humans and, on the other, are completely transparent. That means they can be used to look directly at

cellular processes that give us vital clues about how our own bodies work.

“We started with a simple look and see exercise,” says Feng. They didn't have to wait long to notice something striking – zebrafish larvae develop so quickly, it's like watching cells on fast-forward.

Feng examined zebrafish with healthy cells carrying mutations in cancer-causing genes, known as oncogenes, and tracked how the immune system responded.

“First, we saw that oncogene expression immediately drives an inflammatory response, which attracts certain immune cells to the cell with the mutated oncogene,” Feng explains.

Alongside this inflammation, the mutated cells continued to grow and divide as normal. That was interesting, but it didn’t reveal what those curious immune cells might be doing.

So, the team tried the same experiment in larvae with depleted immune systems. This time, the mutated cells slowed down dividing, and some even died off.

Surprisingly, the immune system, usually tasked with protecting health, seemed to be helping tumours grow.

We don’t see this same immediate inflammatory response to cells with oncogenic mutations in human samples, or even mouse models, but it gives us a window into how promotion can work.

It also raises the possibility that the oncogene-expressing cell type they studied could be tricking our immune cells into supporting tumour initiation, which would be important to explore from a cancer prevention perspective.

“Immune cells are like nurses looking after all the other cell types in your body,” says Feng. “When things are damaged and need to be repaired, they go and help that process.”

The difference with most wounds is the inflammation passes. “The wound heals, the immune cell goes away, and everything goes back to normal,” Feng explains. But when the wounding doesn’t stop; when, for instance, a few rare cigarettes become an addiction, or we’re continually exposed to excessive levels of air pollution, healing can turn into harm.

Promotion in practice

With our funding, Balmain, Feng and others are now working to identify and understand a whole range of different promoters.

That work involves the most advanced technologies – tools that can track a single cell in a field of millions – and it’s only possible thanks to some of the biggest breakthroughs in the history of biology, but it’s pointing to the simplest advice.

"In animal models, we’ve seen that cutting down the frequency and the dose of a promoter can have a disproportionate effect on the total cancer risk,” Balmain says. "It’s much more difficult to prevent mutations, especially as so many of them are happening anyway. But you can prevent promotion by dealing with known promoters in a more sensible manner.”

Those promoters can include alcohol (which can also cause mutations), excessive sugar, and obesity.

“Binge drinking twice a week is not a good idea,” says Balmain. “Having even a small amount every day is probably not a good idea, but promotion is all about the dose and the frequency.

“Your grandmother probably told you what you need to know – what to do and what not to do,” he says. The same advice still applies, even though, on a cellular level, you’re probably not the same person you were when you first heard it.