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

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.

Isn’t Science Wonderful? Obesity Talks to Cancer

A couple of week’s ago we looked at how being obese can give cancer a helping hand. I thought this would be useful as most people know there is a link but perhaps not much more than that. The message had two simple parts: (1) When we make extra fat cells they change the metabolism of our bodies through chemical signals that wander around and, in passing, can also drive cancer growth, and (2) Some of the extra fat cells congregate around tumours and give them direct positive vibes (i.e. other, local chemical signals).

But you may have spotted that I didn’t actually say what these ‘signals’ are – for the very good reason that we know rather little about them. Step forward, right on cue, Ines Barone, Suzanne Fuqua and friends from the University of Calabria and Baylor College of Medicine, Houston with a wonderful paper that’s just been published. Wonderful because it’s got so much data I’m green with envy but also because, like most excellent science papers, the key message is simple: normal cells that have moved into the neighbourhood can indeed talk directly to tumour cells. And the messenger is … leptin!

That’s astonishing. Even those with only a smattering of knowledge about how we work will know that leptin plays a key role in regulating energy balance. It’s a protein – a hormone – that circulates in our blood at levels roughly proportional to body fat. Its job is to signal the ‘full’ state, i.e. to reduce appetite. Somewhat perversely, obesity usually causes abnormally high leptin levels but it doesn’t work very well because the body has become resistant to its signal – much as happens with insulin in type 2 diabetes.

The new results show that leptin, released from nearby cells, can bind to cancer cells and make them do two things: (1) Release a chemical that tells the adjacent cells to send out even more leptin, and (2) Make proteins that help the tumour cells grow and invade.

There are a few wrinkles to these results. The study was on breast cancer cells with a particular mutation (in a receptor for the hormone estrogen) and the ‘groupies’ providing the leptin turned out not to be fat cells but fibroblasts – part of the supportive framework of cells and tissues – so they’re ‘cancer-associated fibroblasts’ (CAFs). And when the CAFs release leptin it floods out and the tumour cells embrace it and make yet more receptors for leptin to bind to on their surface.

But these details matter less than the key point: for at least some types of cancer cell a hormone often made in excessive amounts in obesity can signal directly to tumour cells, telling them to grow and spread. This doesn’t mean that all breast tumours, yet alone all cancers, respond to leptin. What it does show is that a key factor in obesity can talk directly to some types of tumour cell. It’s another example of the painstaking way in which science usually proceeds and, assuming the results are reproducible, we have one more little bit of the jig-saw.

Reference

Barone, I., Catalano, S., Gelsomino, L. et al. (2012). Leptin Mediates Tumor−Stromal Interactions That Promote the Invasive Growth of Breast Cancer Cells. Cancer Research 72, 1416-1427.