Taking a Swiss Army Knife to Cancer

Murder is easy. You just need a weapon and a victim. And, I guess the police would add, opportunity. I hasten to point out that’s an observational note rather than an autobiographical aside. It’s relevant here because treating cancer is intentional homicide on a grand scale – the slaughter of millions of tumour cells. For individuals we cannot say whether the perfect murder is possible – how would we know – but on the mass scale history shows that even the most efficient machines for genocide have been, fortunately, less than perfect. In other words through incredible adaptability, ingenuity, determination and sheer will power, some folk will survive even the most extreme efforts of their fellow men to exterminate them. Cancers tend to mirror their carriers. With only rare exceptions, whatever we throw at them in attempts to eliminate their unwelcome presence, some of the little blighters will dodge the bullets, take a deep breath and start reproducing again. ‘What doesn’t kill you makes you stronger’ as the American chanteuse Kelly Clarkson has it – though to be fair I think she pinched the line from Nietzsche.

Multiple whammys

For cancer cells dodging extinction requires adaptability: using the flexibility of the human genetic code enshrined in DNA to change the pattern of gene expression or to develop new mutations that short-circuit the effects of drugs – finding many different ways to render useless drugs that worked initially. You might draw a parallel with the idea, correct as it turned out, of the prisoners who staged The Great Escape from Stalag Luft III in World War II that if they initially dug three tunnels simultaneously (Tom, Dick and Harry) the guards might find one but they’d probably not find all three.

An approach increasingly permeating cancer therapy is how to target several escape routes at once – can we at least give the tumour cell a serious headache in the hope that while it’s grappling with a molecular carpet bombing it might be more likely to drop dead. One way of doing this is simply to administer drug combinations and this has met with some success. However, for the most part, agents are not specific for tumour cells and their actions on normal cells give rise to the major problem of side effects.

Step forward Yongjun Liu and colleagues from Shandong University, Jinnan, People’s Republic of China and the sophisticated world of chemistry with efforts to fire a broad-shot that combines different ways of killing tumour cells with at least some degree of specific targeting.

Making the bullets

These chemists are clever chaps but, taken one step at a time, what they’ve done to make a very promising agent is simple. The game is molecular Lego – making a series of separate bits then hooking them together. The trendy name is click chemistry, a term coined in 1998 by Barry Sharpless and colleagues at The Scripps Research Institute, to describe reactions in which large, pre-formed molecules are linked to make even more complex multi-functional structures. You could describe proteins as a product of ‘click chemistry’ as cells join amino acid units to make huge chains – but you wouldn’t as it’s better to keep the name for synthetic reactions that make novel modules.

It might help to recall some school chemistry:

acid + base = salt + water (e.g., HCl + NaOH = NaCl + H₂O)

Click chemistry is the same idea but the reactants are large molecules, rather than atoms of hydrogen, chlorine and sodium.

Anti-freeze to anti-cancer in a couple of clicks

The starting point here is remarkably familiar – it’s antifreeze, a chemical added to cooling systems to lower the freezing point of water (e.g., in motor engines). Antifreeze is ethylene glycol (two linked atoms of carbon with hydrogens: HO-CH₂-CH₂-OH): make a string of these molecules and you have a polymer – poly-ethylene glycol (PEG).

For click chemists it’s easy to tag things on to biologicial molecules, including PEG and most proteins. This study used biotin – a vitamin that works like a molecular glue by sticking strongly to another small molecule called avidin, found in egg white. Avidin can therefore be used to fish for anything tagged with biotin – it simply hooks two biotins together. The protein used here is an antibody that binds to a signaling molecule (VEGFR) present on the surface of most tumour cells and blood vessels. VEGFR helps tumour growth by providing a new blood supply – an effect blocked when the antibody binds to it.

Sounds familiar?

If chains of carbon atoms decorated with hydrogens seem familiar, so they should. They’re fats (the saturated fats you get in cream and butter are very similar to the chains of PEG). As anyone who’s done the washing up knows, fats and water don’t get on (which is why we have detergents). Put them in water and fats huddle together in blobs called micelles – sacs of fat. This gives them a useful property: if you mix something else in the water – a drug for instance – and then add PEG and separate the micelles that form, you’ve got drug trapped in a kind of carrier bag. Often called nanoparticles, these small, molecular bubbles made by chemists are packets of drug ready to be delivered.

A sac of poly-ethylene glycol (PEG) with entrapped drug (red dots) tagged in three different ways (Liu et al., 2014).

Addressing the parcel

To turn PEG into a parcel two chemical tricks are needed. The first is to tag PEG with biotin. Now the nanoparticles will pick up VEGFR antibody labeled with avidin – and the antibody label can target the micelles to tumour cells and blood vessels.

Exploding the package

The second trick is the addition of another polymer (a chain of histidine amino acids) that triggers the disassembly of the nanoparticles when they find themselves close to or inside tumour cells – a more acidic environment than the circulation.

Seeing the results

The final twist is to include another modified PEG – this with a chemical group that binds gadolinium when it’s added to the water. Gadolinium is an ion (Gd³⁺) which shows up brightly in MRI scans – the idea being to highlight where the nanoparticles end up after injection into animals.

Does it work?

These multicomponent nanoparticles resemble a Swiss Army knife – all sorts of gadgets sticking out all over the place: PEG to make sacs that contain a drug, biotin hooked to VEGFR antibody to home in on tumour cells, an acidity sensor so the thing falls apart and releases its content on arrival and a contrast enhancer that shows up where this is happening in an MRI scan.

Injected into mice with liver tumours, these multi-functional nanoparticles do indeed home in on the tumours and their surroundings and drastically reduce tumour growth when they carry the drug sorafenib. Sorafenib is the only agent that has been shown to affect liver cancers, although its effects are brief. Compared to sorafenib alone, these new nanoparticles are about three times more potent – presumably because of their targeted delivery.

Where are we?

This wonderfully clever chemistry will not cure liver cancer. A good result when it reaches human trials would be six months remission by comparison with the current average of two months from treatment with sorafenib alone. But what it does show is that hitting cancers hard in multiple ways at least slows them down. We can only hope that more potent drugs and further ingenuity will progressively extend this capacity. The end is not in sight but brilliant technical advances such as that from Yongjun Liu’s lab may be spotlighting the way ahead.

Reference

Yongjun Liu et al., (2014). Multifunctional pH-sensitive polymeric nanoparticles for theranostics evaluated experimentally in cancer. Nanoscale 6, 3231-3242.

Taking the MYC out of cancer

It has been famously said, though no one quite knows who gave first utterance, that England and America are two nations divided by a common language. In deference to readers from the US of A, therefore, we need a word about English before we embark on the current topic. You can get quite a long way in the States on an English accent, partly because the inhabitants are, by and large, very tolerant and cosmopolitan souls and also because they perceive Brits as being rather weird but harmlessly entertaining – at least since they gave up the idea of owning America, signed the Treaty of Paris and slung their hook. But that last phrase is an example of how things can get sticky when you talk to an American audience and it slips your mind that 1783 was quite a long time ago – long enough in fact for a good deal of divergent language evolution. Put another way, use idioms unthinkingly and you can die the death – leaving your listeners wishing that you would indeed sling your hook (American translation: beat it). One of the odder things about this linguistic separation is that American doesn’t have a good phrase that means gently making fun of someone. You can pull a Yankee leg it is true and you may mess them about – but that one’s really fraught as it carries a different innuendo in English. So it’s a pity that over there you can’t extract the Mick, take the Mickey or remove the Michael. It’s deeply regrettable that this phrase probably owes its origins to Cockney rhyming slang referring to the act of urination but strawberries grow in manure and all that. Similar pratfalls work in the other direction, of course, and British audiences are likely to look blank if you mention your keister: start talking about booty and mussing with someone and they’ll really be baffled.

To the current topic. We saw in Mission Impossible that many different pathways pass signals saying ‘grow’ from the outside world to the nucleus at the centre of a cell. Many of these relays use RAS proteins – they’re a major junction in the cellular network so they’re a very tempting target for disruption of signaling. But if all roads lead to Rome, so to speak, is there not an even better target – a main gate, the critical portal through which everything that drives cell proliferation must pass? There is and it’s called MYC (pronounced ‘mick’ – Ah! Now all is clear!!), the gene encoding a protein of the same sound that is a unique master regulator. MYC coordinates the expression of a large panel of genes involved in cell growth and division – it’s essential for cell proliferation.

 

Cell signaling. Many messengers turn on lots of relays that focus on the nucleus telling cells to grow and divide. The MYC protein is a master coordinator.

But there’s an obvious problem: to survive we need to make new cells all the time – about one million every second, just to maintain the status quo. It seems hardly worth pointing out that if you gave someone a drug that blocked MYC it would be fatal: the body simply couldn’t survive for very long with a blocked cell production line. Indeed it’s been known for some time that knocking out the MYC gene in mice is fatal: they fail to develop beyond an early embryonic stage. And yet there’s a huge temptation to ask ‘What would inhibiting MYC do to tumors: might it actually kill them?’ – a curiosity fuelled by the knowledge that MYC is deregulated in most – perhaps all – cancers. That is, an almost invariable upshot of the mutation patterns found in tumors is that excessive amounts of MYC protein are made – and, as it drives cells round the cell cycle and into division, more MYC equals abnormal cell growth, aka cancer.

So, in an experiment that all logic said would not work, Gerard Evan and his colleagues in San Francisco and Cambridge devised a way to switch on an inhibitor of MYC in transgenic mice to ask the unaskable: ‘Is blocking MYC fatal and, if is isn’t, what does it do to tumors?’ The method is to use a trick of genetic engineering to give mice a novel gene that is switched on by dosing them with a drug added to their drinking water. Omitting the drug switches it off. The gene encodes a protein that sticks to the MYC protein and prevents it from interacting with its normal partner so that the dynamic duo that drives cell growth can’t be formed.

The results were startlingly dramatic. First of all, MYC blockade didn’t kill the mice although it certainly had some effects. A shaved patch of fur doesn’t re-grow quickly as it normally would and males become infertile because they can’t make new sperm. But the mice aren’t aware of these little problems and in general are as full of beans as their chums with normal levels of MYC activity – and in any case these mild effects are reversible. Switch off the MYC block and they return rapidly to normal.

That was surprising enough but the really staggering result came from introducing the MYC blockade into mice that develop lung tumors (driven by the expression of a mutant RAS gene). Inhibition of MYC has an almost immediate effect on tumor size: tumors regress and the side effects remain mild and reversible. A single burst of MYC blockade results in a significant extension of life span for tumor-bearing mice. Even more remarkable, successive episodes of MYC inhibition (a ‘metronomic’ regime) leads to the gradual eradication of the tumors – and the elimination of lung cancer means that the mice now have a normal life span.

It should be emphasized that so far this has only happened in mice and the Appian Way of cancer therapy is littered with the corpses of brilliant ideas that worked a treat in those wonderful little models but were utterly useless when it came to humans. However, science is the practice of eternal optimism and there are sound grounds for hope here. Switching a gene on and off by genetic engineering is fine in mice. It won’t do for us but already some small molecule inhibitors have been made that appear to work in mice. The hope is that both the anti-cancer effect and the mild side-effects will be recapitulated in humans – and that, because all pathways do indeed lead to it, taking the MYC out of cancer will kill tumours. Then the really optimistic bit – that even crafty cancer cells will be unable to find a way round such a block. Because all proliferation signals lead to MYC there will be no adaptive mechanism to which cells can turn to ensure their survival.

In short, the tumour will be stuffed – which rather brings us back to where we started except that, having taken the MYC, this needs no translation.

Reference

Soucek, L., Whitfield, J.R., Sodir, N.M., Massó-Vallés, D., Serrano, E., Karnezis, A.N., Swigart, L.B. and Evan, G.I. (2013). Inhibition of Myc family proteins eradicates KRas-driven lung cancer in mice. Genes Dev., 27, 504-513.