Clocking In With Cancer Treatment

I always hesitate to say things like ‘you may recall’ as, from much undergraduate teaching, I’ve learned that blithe throwaways like ‘you’ll remember this from last Monday’s lecture’ tend to be met with blank stares and trying ‘you met this idea in the first year’ on second year classes will draw forth outright mirth blending with mutinous howls. So let’s just start by noting that three weeks ago in Our Inner Self we had a march-past of our intestinal army of bacteria and saw that it is in continuous flux, its make-up oscillating in time to our biological clock – the daily variation that governs most of our bodily functions including the sleep-wake cycle. That’s amazing stuff but a sharp bit of lateral thinking raises interesting questions. If most of the important things in our bodies tick to circadian rhythms, is cell proliferation one of them – after all, the process of cells making more of themselves is at the heart of life. Answer ‘yes.’ But, as abnormal cell proliferation – i.e. something going wrong – is a perfectly adequate three-word definition of cancer, a small step extends the question to ‘do tumours also have rhythm?’ Answer, again, ‘yes.’ A little background before we explain.

Turning back to the clock

In Twenty more winks we saw that there’s a connection between sleep (or rather lack of it) and cancer and showed how two pairs of genes (CRY/PER and CLOCK/BMAL1) lie at the core of circadian timekeeping. They control the sleep-wake cycle and much else. The proteins they make form an orchestrated feedback loop, synchronised by light-induced signalling. That is, the expression of each pair oscillates with a period of roughly 24 hours, but the pairs are out of step to the tune of about 12 hours. The proteins encoded by these genes regulate the expression of many other genes that ensure the cells and tissues of the body beat to an appropriate rhythm. Many messengers spread circadian oscillations around the body via the blood of which, in humans, cortisol (made by the adrenal glands) is perhaps the most familiar (it’s a steroid hormone: the medication dexamethasone is cortisol with two small, extra bits that make it 25 times more potent). You can fairly easily measure cortisol concentration in blood and you’d expect to find that at nine in the morning you’d have roughly double your midnight amount. In other words cortisol is part of your wake-up call. It turns on your appetite, gets you geared up for physical activity and it also activates anti-stress and anti-inflammatory signal pathways. EGFR & cortisol Cross-talk between EGFR and cortisol during the active phase (right: high cortisol) and the resting phase (left: low cortisol). (from Lauriola et al., 2014).

Getting the message across

Taking the memory-prodding risk yet again, in Mission Impossible? we described how biological signals from the outside world bind to receptors (proteins) to convey their message (I’m here, do something!) to the interior of cells. So the picture is: cells receive many signals from messengers that, one way or another, talk to the nucleus, switching on genes that drive proliferation. Most external messengers are proteins themselves – one example is a potent growth promoter called epidermal growth factor (EGF) that works by switching on the EGF receptor (EGFR). Cortisol isn’t a protein: as we’ve noted, it’s a steroid – which means it can diffuse across membranes – but, once inside a cell it works in essentially the same way, by binding to its specific receptor. The upshot of all this is that messengers transmit information from outside the cell to the nucleus – where DNA lives, the cells’ repository of genetic material – so that genes become activated to produce proteins.

Oscillating signals: cellular chattering

The picture of multiple, linear signalling pathways co-existing within cells invites the idea that their protein components might be unable to resist tapping in to their neighbours’ conversations – and so it has turned out. However, for pathways like the EGFR that signal cell growth, cross-talk with cortisol signalling is more than merely listening in. Proteins activated by the steroid hormone can actually interfere with the relays in the EGFR pathway so that EGF signals are suppressed during the active phase (day-time in us, night-time in rodents) but enhanced during the resting phase.

The meaning for life

So growth signaling is under circadian control – by and large our cells do their multiplying when we are at rest. Interesting although perhaps not unexpected. But, as The Bonzo Dog Doo-Dah Band warbled, ‘here comes the twist’ (Urban Spaceman, 1968 if you’re struggling). These pathways are the very ones that are hyper-activated (i.e. mutated) to drive cancer cells to make more of themselves and they are, accordingly, the targets for many anti-cancer drugs. However, chemotherapy is usually administered as single bursts, at daily or longer intervals, and drugs are progressively removed by metabolism thereafter. This means that much of the impact may be lost if, when the drug concentration is at its highest, the target pathway is already suppressed by high glucocorticoids . There’s evidence consistent with this idea from animals bearing EGFR-driven tumours treated with specific inhibitors that are more effective if administered in the resting phase rather than in the active phase.

It’s all in the timing

So two new messages are now making themselves heard in the world of cancer biology. The first is beginning to tell the full story of clock complexity. The second takes up this theme by pointing out that a circadian clock-based model in cancer therapy may offer improved methods for prevention and treatment.

References

Lauriola, M. et al. Diurnal suppression of EGFR signalling by glucocorticoids and implications for tumour progression and treatment. Nat. Commun. 5:5073 doi: 10.1038/ncomms6073 (2014).

Our Inner Self

Richard Gettner is the anti-hero of Christopher Fry’s wonderful play The Dark is Light Enough, set in the Austro-Hungarian war of 1848. Viewing himself as a failed author, failed husband and all-round disaster, he’s just absented himself from the Austrian Army on the basis of not being too nifty at soldiering either. Their minions are hot on his heels, intent on meting out the retribution that the military traditionally reserve for deserters, and he’s taken refuge in the family home of his former wife. In a tête á tête with her she rebukes him for his knack of self-destruction and points out that his book was actually quite well received and wasn’t really a failure. All Gettner’s frustration then bursts forth in a tirade of brutal philosphising:

‘Is there another

Word in the language so unnecessary

As ‘fail’ or ‘failure’?

No one has ever failed to fail in the end;

And for the very evident reason

That we’re made in no fit proportion

To the universal occasion; which, as all

Children, poets and myth-makers know,

Was made to be inhabited

By giants, fiends, and angels of such size

The whole volume of human generations

Could be cupped in their hands;

And very ludicrous it is to see us,

With no more than enough spirit to pray with,

If as much, swarming under gigantic

Stars and spaces.’

Fry deserves to be remembered as one of the great poetic wordsmiths of the English language, if only for The Dark is Light Enough but, had he known that nine out of ten cells in our bodies are bugs, he might have added a final blast to his demolition of the human condition:

Our failings should not surprise as we are but a sinister symbiosis,

More bacterial than human,

Helpfully poised such that when our hour is done

The microbial hordes surge forth to reduce us to our component parts.

bacteria and virus cartoon

The range of the hordes

Our rising preoccupation with the bug army (see it’s a small world & The Best Laid Plans In Mice and Men …) has been promoted by several recent studies that have propelled our ‘inner organism’ from the bowels of biology into the limelight. The story is somewhat fragmented but it’s a good time to see if we can make sense of the current threads.

We’ve known for many years that a motley collection of microorganisms are happy residents in most of our nooks and crannies, ranging from tummy buttons and through the skin, to saliva and our guts. They include bacteria and fungi, they’ve become known as the human microbiome (or microbiota), are said to outnumber human cells 10 to 1 and, all-told, can be viewed as a co-evolved ‘super-organism’ that has many benefits, including making our metabolism more efficient and hence improving nutrition. However, as with everything else in biology, this close relationship is a balancing act, the disturbance of which carries risks for disease development.

It’s critical to note that this vast microbial army, toiling away on our behalf in the dungeon of our innards, mostly dwelling in our gut, is a really mixed lot. It’s estimated to include about 700 different species of bacteria, of which perhaps thirty or forty species make up the bulk. It’s a bit like a mini Great Barrier Reef, well known as the world’s largest coral reef system and extraordinary in that, although it’s made up of billions of tiny organisms, the thing can behave in an integrated way, most dramatically illustrated by mass spawning.

Within the gut there are two major sub-families of microorganisms (Bacteroidetes (Bs) and Firmicutes (Fs)). Although more close-knit genetically speaking, each of these still includes many different classes of microbe. So, they’re a bit of a rabble but, by and large, not only are they harmless, they actually play a vital part in keeping us healthy.

Bacterial army manoeuvres

The power of DNA sequencing means that we can now interrogate our inner armies as to their make up under different conditions, because each type of microbe has a distinctive genome. The first thing to emerge is a dramatic shift in the balance between the major sub-families in obese individuals, be they mice or humans. That is, obese animals have about half the number of Bs and double that of Fs, compared to normal. And the link here is that the bug switch alters the pool of genes available, the upshot being increased energy harvest from nutrients consumed. In other words the switch helps animals get fatter.

It’s possible to breed mice that do not have any gut bugs and ask what happens when you transfer a colony from another animal. Bacteria-free mice on receipt of a normal gut army promptly double body fat: microbiota transferred from obese mice makes ’em twice as fat and, remarkably, human gut microbes from someone who’s obese also makes mice obese, if fed a high-fat rather than a normal diet.

Chemical warfare

Because we use antibiotics on a massive scale to control infections, we might ask whether they cause the good guys to suffer what the military call collateral damage – the point being that antibiotics don’t target bacteria on the basis of whether they’re good for us or potentially fatal. Inevitably, it turns out that ‘good guys’ do get hit by some antibiotics, and when this happens mice gain weight and build up fat. Unsurprisingly, a high-fat diet makes things worse. The sequence is that the drug changes the balance in microbiota before mice become obese and – a real shock – one course of antibiotic treatment imprints these effects on the animal permanently: it acts for life.

To clever for our own good

In our panic to avoid obesity and still pander to our sweet tooth, mankind has taken to using artificial sweeteners on a massive scale in the mistaken belief that these low-calorie agents do no harm. Only recently has this come to light as yet another example of the old adage about there being no such thing as a free lunch. It’s remarkable: saccharin, the most commonly used artificial sweetener, causes big shifts in the proportions of different types of gut bacteria – some increasing whilst others go down – the overall effect again being much more efficient energy harvesting from food. This is a direct effect of saccharin on the bugs, blocked by commonly used antibiotics.

The story so far

The regiments from which our foot soldiers are drawn (i.e. the species that form the microbiota) affect our metabolism and in particular can influence obesity – and that’s inextricably linked with type 2 diabetes and heart disease. With that in mind, it seems obvious that upsetting them with drugs is a risky business. What’s more, seemingly harmless food supplements can also be fraught with danger.

Marching to a beat

Yet another amazing feature of our inner army is that it keeps time. That is, the abundance of different sub-types fluctuates in synchrony with the day/night cycle. Put another way, it marches to a circadian rhythm along with many other physical, mental and behavioral changes that respond mainly to light – and hence roughly follow a 24-hour cycle. These can be big changes in composition: a particular type of bug can double in amount in 6 hours and return to its initial level by 6 hours later. One of the most familiar examples of the importance of biological rhythms comes from upsetting them by flying long distances on an east–west axis. Sure enough, mice have the same problem and, just like us, their clock is disturbed by jet lag (rather than shuttling them business class across the Atlantic you can simulate the effect simply by shifting the light-dark cycle under which they live forwards or backwards by 8 hours every three days). This largely blocks microbiota rhythmicity, the overall effect being to reduce the total number of bacteria. This in turn raises blood sugar level and the mice become obese. These events are absolutely dependent on what has happened to the microbiota because they are replicated in germ-free mice after transfer of jet-lagged faeces.

That’s more astonishing than might appear at first glance because it places the daily variation in gut bug populations alongside the basic circadian rhythms of the sleep-wake cycle, body temperature and other important functions. Circadian rhythms are driven by a ‘master clock’ in the brain that coordinates all the body clocks so that they are in synch. Four proteins are at the heart of the clock (CLOCK and BMAL1, highly expressed during the light phase, and cryptochromes (CRYs) and period proteins (PERs) expressed in the dark phase). These regulate the expression of many genes, thereby controlling the overall response (see Twenty More Winks). The implication is, therefore, that far from being a kind of add-on that occasionally gets upset, our microbiota play central role in a healthy body.

A recent example of it doing just that comes from another mouse model showing our ‘inner organism’ acting to protect against bacteria from the outside world. In response to infection, cells that line the small intestine switch on the production of a particular sugar (fucose): that is then released from the cells and consumed by members of the microbiota – this novel energy source seemingly helping the host to survive the onslaught of infectious microorganisms.

And finally …

All this stuff about germs being our best friends is riveting but what about the important question? Well, there appears to be a complex interaction between diet, microbial metabolism and colorectal cancer, with bacteria able to make some agents that protect against cancer and some others that drive carcinogenesis. There’s evidence that a wide range of tumours can be promoted by transferring microbiota to germ-free mice and, on the other hand, that depleting intestinal bacteria reduces the development of liver and colon cancers.

Space invaders

Personal space is, apparently, a big thing for many of us these days. So big that ‘scientists’ have had a go at measuring it – they never miss an opportunity do they? Actually, boffins being boffins, they measured something called the defensive peripersonal space (DPPS) – a ‘vital safety margin surrounding the body’ – by sticking a pair of electrodes to the wrists of volunteers who held their hands different distances from their faces whilst receiving bursts of current through the electrodes. That made them blink (!) and the nearer the hand to the face the more they blinked, as the shock was perceived to be a greater threat to their face. There is, seemingly, a sharp boundary: up to somewhere between 20 cm and 40 cm is a high-risk area where we get very aerated: beyond that we don’t much care – with large personal variations depending on how twitchy you are. Debrett’s, which styles itself as the arbiter of society etiquette, has a simpler test, its distilled wisdom revealing that if you can feel the warmth of someone’s anxious breath upon your face, then you’re standing too close.

With all this neurosis it’s probably a good job no one mentioned our inner army: a ten-to-one cellular takeover (albeit that bugs are much smaller) is not so much a bit of heavy breathing as a blitzkrieg. Even so, it’s a delicately poised occupation upon which we depend for survival – and it’s one that we disturb at our peril.

References

Sambo, C.F. and Iannetti, G.D. (2013). Better Safe Than Sorry? The Safety Margin Surrounding the Body Is Increased by Anxiety. The Journal of Neuroscience 33, 14225-14230; doi: 10.1523/JNEUROSCI.0706-13.2013.

Twenty more winks

In Episode One we alerted ourselves to the large amount of evidence saying that a good night’s sleep really is essential if you wish to reduce your chances of a wide variety of medical misfortunes. But what do we know about how molecules respond to sleep disruption to produce such nasty effects?

Molecular Clocks

Life on earth depends on energy sent forth by the sun and, in synchrony with the rotation of our planet, many of the inner workings of mammals fluctuate over each period of roughly 24 hours. This pattern is called the circadian clock, its most obvious manifestation being the sleep-wake cycle. Over the years considerable evidence has accumulated that the link between shift-work and cancer is probably due to circadian rhythm disruption and suppression of nocturnal production of a hormone called melatonin. All living things make melatonin (in mammals in the pineal gland of the brain) and it signals through a variety of protein receptors on cells to regulate the sleep-wake cycle but it also plays a role in protecting DNA from damage.

Melatonin production is regulated by the circadian oscillator, itself controlled by two sets of proteins that control each other’s expression in a feedback loop. Thus one pair, CLOCK and BMAL1, activates Cryptochrome and Period. They in turn repress CLOCK and BMAL1 – the upshot being that the activities of both pairs oscillate over a day-night cycle: as one goes up the other comes down. These central regulators are encoded by evolutionarily ancient genes (two for Cryptochromes and three for Period proteins). In plants and insects CRY1 responds to light but in mammals CRY1 and CRY2 work independently of light to inhibit BMAL1-CLOCK.

Two interlocked feedback loops control clock protein expression

CRY-CLOCK

OUTCOME: ≈ 24 hour cycle expression of PER & CRY

BMAL1 & CLOCK 12 hours out of phase

Alarming the Clock

So having sounded the alarm that just one night’s sleep shortage has obvious effects, what do the genes make of it? Well, the short answer is they get upset. A recent study took blood samples from a group of normal people and found that more than 700 genes (about 3% of our total number) significantly changed their level of expression over 1 week of insufficient sleep (5.7 h) by comparison with 1 week of sufficient sleep (8.5 h). About two-thirds were reduced whilst one-third was up-regulated (made more of their protein product). Unsurprisingly, among those that went down were the major clock regulators. It’s worth noting that the sleep perturbation in this experiment was relatively mild – intended to be similar to that experienced by many individuals. The genes most strongly affected play roles in a wide range of biological processes – DNA structure (hence gene expression), metabolism, stress responses and inflammation. The responses of genes to changes in sleep patterns are not the result of mutation (i.e. changes in the sequence of DNA)  but, at least in part, they’re caused by small changes in the structure of DNA. {These are epigenetic modifications – any modification of DNA, other than in the sequence of bases, that affects how an organism develops or functions. They’re brought about by tacking small chemical groups either on to some of the bases in DNA itself or on to the proteins (histones) that act like cotton reels around which DNA wraps itself}. Thus there is evidence for gene silencing by hyper-methylation of CRY2 (adding methyl groups (CH3) to its DNA) and the converse effect of hypo-methylation (removing methyl groups) of CLOCK occurs in women engaged in long-term shift work and is associated with an increased risk of breast cancer.

Inflaming the Problem

The cells that mediate inflammation and immune responses also have circadian clocks – meaning that normally these processes are rhythmically controlled and clock disruption (for example by sleep loss) affects this pattern. Disabling the clock in mice (by knocking out CRY altogether) switches on the release of pro-inflammatory messengers and knocking out one of the Period genes (PER2) makes mice cancer-prone – reflecting the fact that MYC (the key proliferation driver) is directly controlled by circadian regulators and is consistently elevated in the absence of PER2.

Clock Faces

The mass that comprises a tumour is a mixture of cells – cancer cells and normal cells attracted to the locale – so it’s a quite abnormal environment and in particular there may be regions where the supply of oxygen and nutrients is limited. This is sensed as a stress by the cells, one response being to lower protein production until normal conditions are restored. If this doesn’t happen within a given time the response switches to one leading to cell suicide. One way in which overall protein output can be reduced is by activating an enzyme (IRE1α) that breaks down code-carrying messenger RNAs that direct assembly of new proteins. Remarkably, it has emerged that one of the mRNAs targetted by IRE1α is the core circadian clock gene, PER1. The degradation of PER1 mRNA means that less PER1 protein is made, which in turn disrupts the clock. However, it seems that PER1 has other roles that include helping the cell suicide response – a major anti-cancer defence. All of which suggests that disruption of the IRE1α/ PER1 balance might have serious consequences. Indeed IRE1α mutations have been found in a variety of cancers including brain tumours in which low levels of PER1 are an indicator of poor prognosis. The IRE1α mechanism coincidentally activates the transcription factor XBP1 (as well as PER1 mRNA decay) and one target of XBP1 is the gene encoding a messenger (CXCL3) that makes blood vessels sprout offshoots. Thus this master regulator suppresses cell death, activates proliferation (lowering PER1 deregulates MYC) and promotes new blood vessel formation.

A Tip for Snoozing

If you’re still wide awake it just goes to prove the utter fascination of biology – but today’s story says that you have to find ways of, if not falling asleep, at least courting insensibility (as Christopher Fry put it). If it’s a real problem for you may I make a really radical suggestion? Turn to our physicist friends and select from their recent literary avalanche. A ‘brief history of …’ something or other will do fine. It’s a knock-out! Sweet dreams!!

References

Möller-Levet, C.S., Archer, S.N., Bucca, G., Laing, E.E., Slak, A., Kabiljo, R., Lo, J.C.Y., Santhi, N., von Schantz, M., Smith, C.P. and Dijk, D.-J. (2013). Effects of insufficient sleep on circadian rhythmicity and expression amplitude of the human blood transcriptome. PNAS 110, E1132-E1141.

Fu, L.N. et al. (2002). The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111, 41-50.

Zhu, Y. et al. (2011). Epigenetic impact of long-term shiftwork: pilot evidence from circadian genes and whole-genome methylation analysis. Chronobiol Int, 28, 852–861.

Pluquet, O. et al. (2013). Posttranscriptional Regulation of PER1 Underlies the Oncogenic Function of IREα. Cancer Res., 73, 4732-4743.