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.

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Lethal ZIP codes

In Keeping Cancer Catatonic we retailed how, over 125 years ago, the London physician Stephen Paget came up with his ‘seed and soil’ idea to explain why it was that when cancers spread to distant sites around the body by getting into the circulation they didn’t simply stick to the first tissue they came across. Paget had spotted that cancers tend to have preferred sites for spreading: tumours of the eye tend to travel to the liver, rather than the much handier brain, and breast cancers, Paget’s speciality, commonly spread to the liver but also to the lungs, kidneys, spleen and bone. So his idea was that certain distant secondary sites are somehow made more receptive to tumor growth, just as soil can be prepared for seeds to sprout.

So the key question became ‘how?’ and it’s hung in the cancer air for well over a century during which we’ve made very little progress towards an answer – and it is crucial because the business of tumour cells spreading (metastasizing) causes most cancer deaths (over 90%).

But, at long last, things have started to move, largely due to the efforts of David Lyden and his colleagues at Weill Cornell Medical College. Their first astonishing contribution was to show that cells in primary tumours release messengers into the circulation and these, in effect, tag what will become landing points for wandering tumour cells – i.e., the target sites are determined before any tumour cells actually set foot outside the confines of the primary tumour.

After that seismic revelation the story advanced a step further (in Scattering the Bad Seed) with some molecular detail of how the sites are marked – an effect Lyden has christened ‘Bookmarking cancer’ – and how when tumour cells do settle in their new niche they may be kept dormant for many years before starting to expand.

Carrying the flag

The next chapter in the story, as retailed in Holiday Reading (4) – Can We Make Resistance Futile?, revealed that the message is carried by small sacs – like little cells – called exosomes that are released from tumour cells. These float around the circulation until they find their target site, whereupon they plant the flag by setting off a chain reaction that produces a sticky protein – fibronectin – a kind of glue for immune cells and tumour cells.

That is all truly amazing stuff but, as we noted in Holiday Reading (4) – Can We Make Resistance Futile?, a recurring theme in science is that one answer merely poses the next question – in this case ‘what’s the messenger?’

As in all the best thrillers, the authors have kept us in suspense to the last, helped presumably by their not knowing the answer. But in this week’s Nature (Oct. 28, 2015) comes the denoument to this whodunit.

Mister postman look and see …

Many moons ago an outfit called the Marvelettes had a No. 1 hit with Please Mr. Postman and somewhat later the Fab Four did a re-hash that met with equal success. Perhaps we should have asked them how nature would go about directing little packages around the body. John, Ringo and the lads would, with their earthy, Liverpudlian logic, have pointed out the triviality of the problem of exosome addressing. ‘It’s not like you’re sending stuff all over the world, is it? You’ve only got a few targets – the major organs of the body. So a dead simple code will do. You know your messengers are proteins – ’coz they do everything – OK? So, pick a protein that comes in two bits with a few variants of each: mix and match and there’s yer postcodes. Now … what was that ditty about yellow subsurface vessels …’

And so it came to pass …

And the messenger is …

A family of proteins called integrins whose job is to span the membranes of cells, thereby promoting cell-cell interactions. They are indeed made of two different chains stuck together (called α (alpha) and β (beta)) and the upshot is that our cells can make about 24 unique integrins – more than enough to form a coded address system to direct tumour cells around the body. Well done lads!

What Ayuko Hoshino, David Lyden and their many collaborators did was to tag exosomes released from various types of cancer cell with a fluorescent dye and inject them into mice. The fluorescent label enabled them to track the exosomes and it turned out that, for a variety of cancer cells (breast, pancreatic, colorectal, lung, melanoma and pediatric) the exosomes travelled to the organs associated with metastasis (e.g., breast cancer exosomes stuck in the lungs, pancreatic cancer exosomes in the liver, etc). In other words exosome spread mimicked the pattern of the tumour from which they were derived. Once they had landed the exosomes set about reprogramming the organ sites to make a fertile microenvironment capable of supporting tumor cell growth in a new colony.

When they looked at the exosome proteins they found a particular member of the integrin family flagged each organ-specific site. Thus α6β4 promotes lung metastasis, αvβ5 homes in on the liver, αvβ3 on the brain, etc.

MapFinding a home

To spread around the body (metastasise) primary tumours first release small sacs (exosomes) carrying protein tags (integrins). Moving through the circulatory system the integrin tags home in to specific addresses found on different organs. The effect of exosomes sticking to target sites is to prepare the ground for cells released by the tumour to adhere and colonise.

Down the tube

You could think of primary tumours as being a bit like us when we move to a new city and try to find a des. res. in a place you don’t know. We could just ramble round the subway system until something catches our eye but that might take for ever. Much more efficient is to ask someone with local knowledge where would be good spots to target. For disseminating tumours their exosomes are the scouts who do the foot-slogging: the protein signatures on the surface of these small, tumour-secreted packages home in on postcodes that define a desirable locale for metastatic spread.

Shooting the messenger

An obvious question is ‘If exosomes are critical in defining metastatic sites, can you block their action – and what happens when you do?’ In preliminary experiments Hoshino & Co showed that either knockdown of specific integrins or blocking the capacity of these proteins to stick to their targets (with a specific antibody or short synthetic peptides) significantly reduced exosome adhesion, thereby blocking pre-metastatic niche formation and liver metastasis.

A new beginning?

We described these fabulous results as the denouement but, of course, it isn’t. As Mr. Churchill remarked in a somewhat different context: ‘Now this is not the end.’ It is rather a step to answering an old question but it’s incredibly exciting. If screening for exosomes leads to the detection of cancer not just years but perhaps decades earlier than can be achieved by present methods and if blocking their action can keep metastasis at bay, then the field of cancer will be utterly transformed.

References

Hoshino, A. et al. (2015). Tumour exosome integrins determine organotropic metastasis. Nature doi:10.1038/nature15756.

Ruoslahti, E. (1996). RGD and Other Recognition Sequences for Integrins. Annual Review of Cell and Developmental Biology 12, 697-715.