Going With The Flow

The next time you happen to be in Paris and have a spare moment you might wander over to, or even up, the Eiffel Tower. The exercise will do you good, assuming you don’t have a heart attack, and you can extend your knowledge of science by scanning the names of 72 French scientists that you’ll find beneath the square thing that looks like a 1st floor balcony. Chances are you won’t recognize any of them: they really are History Boys – only two were still alive when Gustave Eiffel’s exhibit was opened for the 1889 World’s Fair.

One of the army of unknowns is a certain Michel Eugène Chevreul – and he’s a notable unknown in that he gave us the name of what is today perhaps the most familiar biological chemical – after DNA, of course. Although Chevreul came up with the name (in 1815) it was another Frenchman, François Poulletier de la Salle who, in 1769, first extracted the stuff from gallstones.

A few clues

The ‘stuff’ has turned out to be essential for all animal life. It’s present in most of the foods we eat (apart from fruit and nuts) and it’s so important that we actually make about one gram of it every day to keep up our total of some 35 grams – mostly to be found in cell membranes and particularly in the plasma membrane, the outer envelope that forms the boundary of each cell. The cell membrane protects the cell from the outside world but it also has to allow chemicals to get in and out and to permit receptor proteins to transmit signals across the barrier. For this it needs to be flexible – which why membranes are formed from two layers of lipids back-to-back. The lipid molecules have two bits: a head that likes to be in contact with water (blue blobs in picture) to which is attached two ‘tails’ ­– fatty acid chains (fatty acids are unbranched chains of carbon atoms with a methyl group (CH3–) at one end and a carboxyl group (–COOH) at the other).

Bilayer

Cholesterol_molecule_ball

A lipid bilayer                                          

De la Salle’s substance

 

The lipid ‘tails’ can waggle around, giving the plasma membrane its fluid nature and, to balance this, membranes contain roughly one molecule of ‘stuff’ for every lipid (the yellow strands in the lipid bilayer). As you can see from the model of the substance found by de la Salle, it has four carbon rings with a short, fatty acid-like tail (the red blob is an oxygen atom). This enables it to slot in between the lipid tails, strengthening the plasma membrane by making it a bit more rigid, so it’s harder for small molecules to get across unless there is a specific protein carrier.

Bilayer aThe plasma membrane. A fluid bilayer made of phospholipids and cholesterol permits proteins to diffuse within the membrane and allows flexibility in their 3D structures so that they can transport small molecules and respond to extracellular signals.


De la Salle’s ‘stuff’ has become famous because high levels have been associated with heart disease and much effort has gone into producing and promoting drugs that reduce its level in the blood. This despite the fact that numerous studies have shown that lowering the amount of ‘stuff’ in our blood has little effect on mortality. In fact, if you want to avoid cardiovascular problems it’s clear your best bet is to eat a Mediterranean diet (mostly plant-based foods) that will make no impact on your circulating levels of ‘stuff’.

By now you will have worked out that the name Chevreul came up with all those years ago is cholesterol and it will probably have occurred to you that it’s pretty obvious that our efforts to tinker with it are doomed to failure.

We’ve known for along time that if you eat lots of cholesterol it doesn’t make much difference to how much there is in your bloodstream – mainly because cholesterol in foods is poorly absorbed. What’s more, because it’s so important, any changes we try to make in cholesterol levels are compensated for by alterations the internal production system.

Given how important it is and the fact that we both eat and make cholesterol, it’s not surprising that quite complicated systems have evolved for carting it around the body and delivering it to the right places. These involve what you might think of as molecular container ships: called lipoproteins they are large complexes of lipids (including cholesterol) held together by proteins. The cholesterol they carry comes in two forms: cholesterol itself and cholesterol esters formed by adding a fatty acid chain to one end of the molecule – which makes them less soluble in water.

lipoprotein-structureChol est fig

Lipoprotein                                                               Cholesterol ester

Formed by an enzyme – ACAT –
adding a fatty acid to cholesterol.
Avasimibe blocks this step.

 

So famous has cholesterol become even its taxi service has passed into common language – almost everyone knows that high-density lipoproteins (HDLs) carry so-called ‘good cholesterol’ (back to the liver for catabolism) – low concentrations of these are associated with a higher risk of atherosclerosis. On the other hand, high concentrations of low-density lipoproteins (LDLs) go with increasing severity of cardiovascular disease – so LDLs are ‘bad cholesterol’.

What’s this got to do with cancer?

The evidence that cholesterol levels play a role in cancer is conflicting. A number of studies report an association between raised blood cholesterol level and various types of cancer, whilst others indicate no association or the converse – that low cholesterol levels are linked to cancers. However, the Cancer Genome Atlas (TCGA) that profiles DNA mutations and protein expression reveals that the activity of some genes involved in cholesterol synthesis reflect patient survival for some cancer types: increased cholesterol synthesis correlating with decreased survival. Perhaps that accounts for evidence that the class of cholesterol lowering drugs called statins can have anti-tumour effects.

In a recent development Wei Yang and colleagues from various centres in China have come up with a trick that links cholesterol metabolism to cancer immunotherapy. They used a drug (avasimibe) that blocks the activity of the enzyme that converts cholesterol to cholesterol ester (that’s acetyl-CoA acetyltransferase – ACAT1). The effect of the drug is to raise cholesterol levels in cell membranes, in particular, in killer T cells. As we’ve noted, this will make the membranes a bit more rigid and a side-effect of that is to drive T cell receptors into clusters.

One or two other things happen but the upshot is that the killer T cells interact more effectively with target tumour cells and toxin release by the T cells – and hence tumour cell killing – is more efficient. The anti-cancer immune response has been boosted.

Remarkably, it turned out that when mice were genetically modified so that their T cells lacked ACAT1, a subset of these cells (CD8+) up-regulated their cholesterol synthesis machinery. Whilst this seems a paradoxical response, it’s very handy because it is these CD8+ cells that kill tumour cells. Avasimibe has been shown to be safe for short-term use in humans but the genetic engineering experiments in mice suggest that a similar approach, knocking out ACAT1, could be used in human immunotherapy.

References

Yang, W. et al. (2016). Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism. Nature 531, 651–655.

Dustin, M.L. (2016). Cancer immunotherapy: Killers on sterols. Nature 531, 583–584.

 

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Where’s that Tumour?

It’s handy that in the last piece we summarised the Grand Plan of President Obama’s Moonshot and the UK’s complementary Grand Challenges for cancer because it’s a good backdrop to some results presented a month ago at the European Breast Cancer Conference in Amsterdam. As ever, the newspapers reported them under ‘staggering’ headlines – but this time you couldn’t really blame them as one of the boffins involved, Nigel Bundred of Manchester University, described the results as mind-boggling.’

Prepare to be boggled

What was reported was a small-scale trial (257 women) of a treatment for one of the most aggressive forms of breast cancer – HER2 positive. This subtype of breast cancer takes its name from a protein that spans the cell membrane and can pass a signal from outside to in. That makes HER2 a ‘receptor’ – you can think of receptors as two blobs of protein joined by a wiggly bit that sits across the cell membrane. When something sticks to the outer bit the receptor changes shape to accommodate it. It’s rather like shaking hands with someone: the shape of your hand changes as you grip theirs. The clever bit is that a relatively small change in the blob on the outside of the cell is transmitted to the blob on the inside via the trans-membrane bridge (or wiggly bit).

HER2 is unusual: rather than having its own messenger floating around in the circulation, it gets switched on by sticking to another cell surface receptor – such receptors are rather touchingly called ‘orphans’. HER2 is a bit of an incestuous orphan, being particularly fond of HER3, a close relative – and when these two are drawn into an embrace on the outside of the cell their internal blobs have to follow suit – it’s difficult to kiss while keeping your bottom halves far apart. This drawing together of the internal blobs in turn causes them to change shape – not a lot but just enough to act as a signal. For HER2 that signal is an enzyme activity: it gets turned on as a kinase – so it adds phosphate groups, specifically to tyrosine amino acids, in target proteins. It’s a receptor tyrosine kinase. Switching it on activates downstream pathways that signal to the nucleus, telling the cell to go forth and multiply.

Because there are lots of signal pathways in cells that send messages in straight lines but can also ‘cross-talk’, it’s a bit like a blancmange: poke it in one place with a chemical (messenger or drug) and the whole thing wobbles.

Fig. 1. 114

The cell as a blancmange. Receptor proteins span the outer membrane and most pass a signal from outside to in as a response to the arrival of a chemical messenger. HER2 is unusual because it works by linking with other receptors (e.g. HER3): the intracellular pathways thus activated include RAS-MAPK.

Healthy breast cells have about 20,000 HER2 proteins but tumour cells may have 100 times more – i.e. 2 million receptors. So it’s easy to see that if you jack up the number of signallers by 100-fold you’re likely to have a pretty hefty proliferation push. The cells just keep on making more and more of themselves in an uncontrolled way – that’s cancer.

One of the main downstream signalling pathways from HER2 is RAS-MAPK that we’ve met before as a seductive target for blocking by anti-cancer drugs.

But, because multiple pathways can be switched on, hitting a single target often doesn’t work too well.

What’s new?

The usual treatment for breast cancer is primary tumour removal by surgery followed by a combination of radiotherapy and drugs. One of the most successful drugs for treating cancers with high levels of HER2 has been trastuzumab (brandname Herceptin). Herceptin is an antibody that sticks to HER2, prevents the receptor interacting with other proteins (including HER3) and thus blocks uncontrolled signalling.

The study that’s just been reported had two novel twists. The first was to try Herceptin before surgery. The second was to combine Herceptin with another drug – one that hits the enzyme activity that turns on the signal pathways inside cells.

A big turn-off: kinase inhibitors

Lapatinib (Tykerb/Tyverb) is a small molecule that inhibits the tyrosine kinase activity of HER2. It’s been used hitherto where a cancer has progressed after treatment with other drugs. About a dozen kinase inhibitors currently have Food and Drug Administration approval with many more in clinical trials. Perhaps the best known is imatinib (Gleevec), used for the treatment of chronic myelogenous leukemia.

Combining Tykerb with Herceptin hits the signal pathway two different spots. The idea is to give the tumour cell two problems to overcome in the hope that it will fail. It’s a strategy that has met with some success in other settings – meaning that some patients have had extended survival times.

In this study 66 women were given the combination therapy and the results clearly came as a serious shock to one and all. In almost nine out of ten cases there was an immediate response but in 11% tumours entirely vanished over a two-week treatment period. That is truly astonishing. Even in the most successful mouse experiments it is a very rare event for tumours to disappear. In a further 17% of the women tumours shrunk to less than 5mm – a growth so small it is classed as “minimal residual disease”.

Fig. 2. 114

Poking the blancmange. Two shots at blocking signalling in a cancer cell with high levels of the HER2 receptor. Herceptin prevents HER2 interacting with other proteins, especially HER3, whilst Tykerb blocks any residual tyrosine kinase activity.

 A big question, of course, is why complete responses only occurred in one in ten cases – and it underlines the need to know more about what makes a tumour, as we noted last time. That aside, one very encouraging aspect is the short treatment period required for a response. Tyverb was turned down by NHS rationing bodies for not being cost-effective at £27,000 a year – much the same as Herceptin. However, the combined therapy would be about £1,500 per patient. Assuming that the complete responders really are in long-term remission, that would represent a financial transformation almost as astonishing as the biological result.