What’s New in Breast Cancers?

 

One of the best-known things about cancer is that it’s good to catch it early. By that, of course, we don’t mean that you should make an effort to get cancer when you’re young but that, if it does arise it’s a good idea to find out before the initial growth has spread to other places in the body. That’s because surgery and drug treatments are very effective at dealing with ‘primary’ tumours — so much so that over 90% of cancer deaths are caused by cells wandering away from primaries to form secondary growths — a process called metastasis — that are very difficult to treat.

The importance of tumour spreading is shown by the figures for 5-year survival rates. Overall in the USA it’s 90% but this figure falls to below 30% for cancers that have metastasized (e.g., to the lungs, liver or bones). For breast cancer the 5-year survival rate is 99% if it is first detected only in the breast (most cases (62%) are diagnosed at this stage). If it’s spread to blood and lymph vessels in the breast the 5-year survival rate is 85%, dropping to 27% if it’s reached distant parts of the body.

What’s the cause of the problem?

The other thing most people know about cancers is that they’re caused by damage to our genetic material — DNA — that is, by mutations. This raises the obvious notion that secondary tumours might be difficult to deal with because they have accumulated extra mutations compared with those in primaries. And indeed, there have been several studies pointing to just that.

Very recently, however, François Bertucci, Fabrice André and their colleagues in various institutes in France, Switzerland and the USA have mapped in detail the critical alterations in DNA that accumulate as different types of breast cancers develop from early tumours to late, metastatic forms. As is the way these days, their paper contains masses of data but the easiest form of the message comes in the shape of ‘violin plots’. These show the spread of results  — in this case the number of mutations per length of DNA.

Metastatic tumours have a bigger mutational load than early tumours. These plots are for one type of breast tumour (HR+/HER2−) and show results for 381 metastases and 501 early tumours. Red dots = median values: these are the “middle” values rather than an average (or mean) and they show a clear upwards shift in burden as early tumours evolve into metastases. From Bertucci et al., 2019.

The violin plots above are for one subtype of breast cancer (HR+/HER2−). Recall that breast tumours are often defined by which of three types of protein can be detected on the surface of the cells: these are ‘receptors’ that have binding sites for the hormones estrogen and progesterone and for human epidermal growth factor. Hence they are denoted as hormone receptors (HRs) and (human) epidermal growth factor receptor-2 (HER2). Thus tumours may have HRs and HER2 (HR+, HER2+) or various receptors may be undetectable. Triple negative breast cancer (TNBC) is an absence of receptors for both estrogen and progesterone and for HER2.

The plots clearly show an increase in mutation load with progression from early to metastatic tumours (on average from 2.4 to 3.8 mutations per megabase of DNA). Looking at individual genes, nine ‘drivers’ emerged that were more frequently mutated in HR+/HER2− metastatic breast cancers (we described ‘driver’ and ‘passenger’ mutations in Taking Aim at Cancer’s Heart).

So what?

For now these findings give us just a little more insight into what goes on at the molecular level to turn a primary into a metastatic tumour. The fact that some of the acquired driver mutations are associated with poor patient survival offers some guidance as to treatment options.

Don’t get carried away

It’s a familiar story in this field: another small advance in piecing together the jigsaw that is cancer. It doesn’t offer any immediate advance in treatment — mainly because most of the nine ‘driver’ genes identified are tumour suppressors — i.e. they normally act as brakes on cell growth. Mutations knock out that activity and at the moment there is no therapeutic method for reversing such mutations. (The other main class of cancer promoters is ‘oncogenes‘ in which mutations cause hyper-activity).

But such steps are important. The young slave girl in Uncle Tom’s Cabin gave us the phrase “grew like Topsy” — meaning unplanned growth. Cancer growth is indeed unplanned and a bit like Topsy but it’s driven by molecular forces and only through untangling these can we begin to design therapies in a rational way.

Reference

Bertucci, F. et al. (2019). Genomic characterization of metastatic breast cancers. Nature 569, 560–564.

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Caveat Emptor

 

It must be unprecedented for publication of a scientific research paper to make a big impact on a significant sector of the stock market. But, in these days of ‘spin-off’ companies and the promise of unimaginable riches from the application of molecular biology to every facet of medicine and biology, perhaps it was only a matter of time. Well, the time came with a bang this June when the journal Nature Medicine published two papers from different groups describing essentially the same findings. Result: three companies (CRISPR Therapeutics, Editas Medicine and Intellia) lost about 10% of their stock market value.

I should say that a former student of mine, Anthony Davies, who runs the Californian company Dark Horse Consulting Inc., mentioned these papers to me before I’d spotted them.

What on earth had they found that so scared the punters?

Well, they’d looked in some detail at CRISPR/Cas9, a method for specifically altering genes within organisms (that we described in Re-writing the Manual of Life).

Over the last five years it’s become the most widely used form of gene editing (see, e.g., Seeing a New World and Making Movies in DNA) and, as one of the hottest potatoes in science, the subject of fierce feuding over legal rights, who did what and who’s going to get a Nobel Prize. Yes, scientists do squabbling as well as anyone when the stakes are high.

Nifty though CRISPR/Cas9 is, it has not worked well in stem cells — these are the cells that can keep on making more of themselves and can turn themselves in other types of cell (i.e., differentiate — which is why they’re sometimes called pluripotent stem cells). And that’s a bit of a stumbling block because, if you want to correct a genetic disease by replacing a defective gene with one that’s OK, stem cells are a very attractive target.

Robert Ihry and colleagues at the Novartis Institutes for Biomedical Research got over this problem by modifying the Cas9 DNA construct so that it was incorporated into over 80% of stem cells and, moreover, they could switch it on by the addition of a drug. Turning on the enzyme Cas9 to make double-strand breaks in DNA in such a high proportion of cells revealed very clearly that this killed most of them.

When cells start dying the prime suspect is always P53, a so-called tumour suppressor gene, switched on in response to DNA damage. The p53 protein can activate a programme of cell suicide if the DNA cannot be adequately repaired, thereby preventing the propagation of mutations and the development of cancer. Sure enough, Ihry et al. showed that in stem cells a single cut is enough to turn on P53 — in other words, these cells are extremely sensitive to DNA damage.

Gene editing by Cas9 turns on P53 expression. Left: control cells with no activation of double strand DNA breaks; right: P53 expression (green fluorescence) several days after switching on expression of the Cas9 enzyme. Scale bar = 100 micrometers. From Ihry et al., 2018.

In a corresponding study Emma Haapaniemi and colleagues from the Karolinska Institute and the University of Cambridge, using a different type of cell (a mutated line that keeps on proliferating), showed that blocking P53 (hence preventing the damage response) improves the efficiency of genome editing. Good if you want precision genome editing by risky as it leaves the cell vulnerable to tumour-promoting mutations.

Time to buy?!

As ever, “Let the buyer beware” and this certainly isn’t a suggestion that you get on the line to your stockbroker. These results may have hit share prices but they really aren’t a surprise. What would you expect when you charge uninvited into a cell with a molecular bomb — albeit one as smart as CRISPR/Cas9. The cell responds to the DNA damage as it’s evolved to do — and we’ve known for a long time that P53 activation is exquisitely sensitive: one double-strand break in DNA is enough to turn it on. If the damage can’t be repaired P53’s job is to drive the cell to suicide — a perfect system to prevent mutations accumulating that might lead to cancer. The high sensitivity of stem cells may have evolved because they can develop into every type of cell — thus any fault could be very serious for the organism.

It’s nearly 40 years since P53 was discovered but for all the effort (over 45,000 research papers with P53 in the title) we’re still remarkably ignorant of how this “Guardian of the Genome” really works. By comparison gene editing, and CRISPR/Cas9 in particular, is in its infancy. It’s a wonderful technique and it may yet be possible to get round the problem of the DNA damage response. It may even turn out that DNA can be edited without making double strand breaks.

So maybe don’t rush to buy gene therapy shares — or to sell them. As the Harvard geneticist George Church put it “The stock market isn’t a reflection of the future.” Mind you, as a founder of Editas Medicine he’d certainly hope not.

References

Ihry, R.J. et al. (2018). p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nature Medicine, 1–8.

Haapaniemi, E. et al. (2018). CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nature Medicine (2018) 11 June 2018.

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