Food Fix For Pharma Failure

 

If you held a global quiz, Question: “Which biological molecules can you name?” I guess, setting aside ‘DNA‘, the top two would be insulin and glucose. Why might that be? Well, the World Health Organization reckons diabetes is the seventh leading cause of death in the world. The number of people with diabetes has quadrupled in the last 30 years to over 420 million and, together with high levels of blood glucose (sugar), it kills nearly four million a year.

There are two forms of diabetes: in both the level of glucose in the blood is too high. That’s normally regulated by the hormone insulin, made in the pancreas. In Type 1 diabetes insulin isn’t made at all. In Type 2 insulin is made but doesn’t work properly.

When insulin is released into the bloodstream it can ‘talk’ to cells by binding to protein receptors that span cell membranes. Insulin sticks to the outside, the receptor changes shape and that switches on signalling pathways inside the cell. One of these causes transporter molecules to move into the cell membrane so that they can carry glucose from the blood into the cell. When insulin doesn’t work it is this circuit that’s disrupted.

Insulin signalling. Insulin binds to its receptor which transmits a signal across the cell membrane, leading to the activation of the enzyme PIK3. This leads indirectly to the movement of glucose transporter proteins to the cell membrane and influx of glucose.

So the key thing is that, under normal conditions, when the level of blood glucose rises (after eating) insulin is released from the pancreas. Its action (via insulin receptors on target tissues e.g., liver, muscle and fat) promotes glucose uptake and restores normal blood glucose levels. In diabetes, one way or another, this control is compromised.

Global expansion

Across most of the world the incidence of diabetes, obesity and cancer are rising in parallel. In the developed world most people are aware of the link between diabetes and weight: about 90% of adults with diabetes are overweight or obese. Over 2 billion adults (about one third of the world population) are overweight and nearly one third of these (31%) are obese — more than the number who are underweight. The cause and effect here is that obesity promotes long-term inflammation and insulin resistance — leading to Type 2 diabetes.

Including cancer

The first person who seems to have spotted a possible connection between diabetes and cancer was the 19th-century French surgeon Theodore Tuffier. He was a pioneer of lung and heart surgery and of spinal anaesthesia and he’s also a footnote in the history of art by virtue of having once owned A Young Girl Reading, one of the more famous oil paintings produced by the prolific 18th-century artist Jean-Honoré Fragonard (if you want to see it head for the National Gallery of Art in Washington DC). Tuffier noticed that having type 2 diabetes increased the chances of patients getting some forms of cancer and pondered whether there was a relationship between diabetes and cancer.

It was a good question then but it’s an even better one now when this duo have become dominant causes of morbidity and mortality worldwide.

We now know that being overweight increases the risk of a wide range of cancers including two of the most common types — breast and bowel cancers. Unsurprisingly, the evidence is also clear that diabetes (primarily type 2) is associated with increased risk for some cancers (liver, pancreas, endometrium, colon and rectum, breast, bladder).

With all this inter-connecting it’s perhaps not surprising that the pathway by which insulin regulates glucose also talks to signalling cascades involved in cell survival, growth and proliferation — in other words, potential cancer initiators. The central player in all this is a protein called PIK3 (it’s an enzyme that adds phosphate groups (so it’s a ‘kinase’) to a lipid called phosphatidylinositol bisphosphate, an oily, water-soluble component of the plasma membrane). It’s turned out that PIK3 is one of the most commonly mutated genes in human cancers — e.g., PIK3 mutations occur in 25–40% of all human breast cancers.

Signalling pathways switched on by mutant PIK3. A critical upshot is the activation of cell survival and growth that leads to cancer.

Accordingly, much effort has gone into producing drugs to block the action of PIK3 (or other steps in this signal pathway). The problem is that these have worked as cancer treatments either very variably or not at all.

The difficulty arises from the inter-connectivity of signalling that we’ve just described: a drug blocking insulin signalling causes the liver to release glucose and prevents muscle and fats cells from taking up glucose. Result: blood sugar levels rise (hyperglycaemia). This effect is usually transient as the pancreas makes more insulin that restores normal glucose levels.

Blockade of mutant PIK3 by an inhibitor. This blocks the route to cancer but glucose levels rise in the circulation (hyperglycaemia) promoting the release of insulin (top). Insulin can now signal through the normal pathway (bottom), overcoming the effect of the anti-cancer drug. Note that the cell has two copies of the PIK3 gene/protein, one of which is mutated, the other remaining normal.

Is our journey really necessary?

By now you might be wondering whether there is anything that makes grappling with insulin signaling worth the bother. Well, there is — and here it is. It’s a recent piece of work by Benjamin Hopkins, Lewis Cantley and colleagues at Weill Cornell Medicine, New York who looked at ways of getting round the insulin feedback response so that the effect of PIK3 inhibitors could be boosted.

Sketch showing the effect of diet on the potency of an anti-cancer drug in mice. The red line represents normal tumour growth. The black line shows the effect of PIK3 blockade when the mice are on a ketogenic diet: tumour growth is suppressed. On a normal diet the drug alone has only a slight effect on tumour growth. Similar results were obtained in a variety of model tumours (Hopkins et al., 2018).

They first showed that, in a range of model tumours in mice, insulin feedback caused by blockade of PIK3 was sufficient to switch on signalling even in the continued presence of anti-PIK3 drugs. The really brilliant result was that changing the diet of the mice could offset this effect. Switching the mice to a high-fat, adequate-protein, low-carbohydrate (sugar) diet essentially stopped the growth of tumours driven by mutant PIK3 treated with PIK3 blockers. This is a ketogenic (or keto) diet, the idea being to deplete the store of glucose in the liver and hence limit the rise in blood glucose following PIK3 blockade.

Giving the mice insulin after the drug drastically reduces the effect of the PIK3 inhibitor, supporting the idea that that a keto diet improves responses to PIK3 inhibitors by reducing blood insulin and hence its capacity to switch on signalling in tumour cells.

A few weeks prior to the publication of the PIK3 results another piece of work showed that adding the amino acid histidine to the diet of mice can increase the effectiveness of the drug methotrexate against leukemia. Methotrexate was one of the first anti-cancer agents to be made and has been in use for 70 years.

These are really remarkable results — as far as I know the first time diet has been shown to influence the efficacy of anti-cancer drugs. It doesn’t mean that all tumours with mutations in PIK3 have suddenly become curable or that the long-serving methotrexate is going to turn out to be a panacea after all — but it does suggest a way of improving the treatment of many types of tumour.

References

Hopkins, B.D. et al. (2018). Suppression of insulin feedback enhances the efficacy of PI3K inhibitors. Nature 560, 499-503.

Kanarek, N. et al. (2018). Histidine catabolism is a major determinant of methotrexate sensitivity. Nature 559, 632–636.

Now You See It

 

In the pages of this blog we’ve often highlighted the power of fluorescent tags to track molecules and see what they’re up to. It’s a method largely pioneered by the late Roger Tsien and it has revolutionized cell biology over the last 20 years.

In parallel with molecular tagging has come genetic engineering that permits novel genes, usually carried by viruses, to be introduced to cells and animals. As we saw in Gosh! Wonderful GOSH and Blowing Up Cancer, various ‘virotherapy’ approaches have been used with some success to treat leukemias and skin cancers and a trial is underway in China treating metastatic non-small cell lung cancer.

A major aim of genetic engineering is to be able to control the expression of novel genes (i.e. protein production from the encoding DNA sequence) that have been introduced into an animal — in the jargon, to ‘switch’ on or off at will. That can be done but only by administering a drug or some other regulator, either in drinking water, by injection or squirting directly into the lungs. An ideal would be something that’s more controlled and less invasive. How about shining a light on the relevant spot?!

Wacky or what?

That may sound as though we’re veering towards science fiction but reflect for a moment that every animal with vision, however rudimentary, sees by transforming light entering the eyes into electrical signals that the brain turns into a picture of the world around them. This relies on photoreceptor proteins that span the membranes of retinal cells.

How vision works. Light passes through the lens and falls on the retina at the back of the eye. The photoreceptor cells it activates are rod cells (that respond to low light levels — there’s about 100 million of them) and cone cells (stimulated by bright light). Sitting across the membranes of these cells are photoreceptor proteins — rhodopsin in rods and photopsin in cones. Photoreceptor proteins change shape when light falls on them — the driver for this being a small chemical attached to the proteins called retinal, one of the many forms of vitamin A. This shape change allows the proteins to ‘talk’ to the inside of the cell, i.e. to interact with other proteins to switch on enzymes and change the level of ions (sodium and calcium). The upshot is that the signal is passed through neural cells in the optic nerve to the brain where the incoming light signals are processed into the images that we perceive. © Arizona Board of Regents / ASU Ask A Biologist.

The seemingly far-fetched notion of controlling genes by light was floated by Francis Crick in 1999. The field was launched in 2002 by Boris Zemelman and Gero Miesenböck who engineered neurons to express one form of rhodopsin. This gave birth to the subject of optogenetics — using light to control cells in living tissues that have been genetically modified to express light-sensitive ion channels such as rhodopsin. By 2010 optogenetics had advanced to being the ‘Method of the Year’ according to the research journal Nature Methods.

Dropping like flies

One of the most dramatic demonstrations of the power of optogenetics has come from Robert Kittel and colleagues in Würzburg and Göttingen who made a mutant form of a protein called channelrhodopsin-1 (found in green algae) and expressed it in fruit flies (Drosophila melanogaster). The mutant protein (ChR2-XXL) carries very large photocurrents of ions (critically sodium and calcium) with the result that photostimulation can drastically change the behaviour of freely moving flies.

Light-induced stimulation of motor neurons in adult flies expressing a mutant form of rhodopsin ChR2-XXL. Click to run movie.

Left hand tube: Activation of ChR2-XXL in motor neurons with white light LEDs caused reversible immobilization of adult flies. In contrast (right hand tube) flies expressing normal (wild-type) channelrhodopsin-2 showed no response. From Dawydow et al., 2014.

Other optogenetic experiments on flies can be viewed on You Tube, e.g., the TED talk of Gero Miesenböck and the Manchester Fly Facility video of fly maggots, engineered to have a channel protein (channelrhodopsin) in their neurons, responding to blue light.

Of flies … and mice … and men

This is stunning science and it’s opened a new vista in neurobiology. But what about the things we’re concerned with in these pages — treating diseases like diabetes and cancer?

Scheme showing how genetic engineering can make the release of insulin from cells controllable by light. Normally cells of the pancreas (beta cells) take up glucose when its level in the circulation rises (via a glucose transporter protein). The rise in glucose triggers ATP production in the cell. This in turn causes potassium channels in the membrane to close (called depolarization) and this opens calcium channels. The increase in calcium in the cell drives insulin secretion. From Kushibiki et al., 2015.

The left-hand scheme above shows how glucose triggers the pancreas to produce the hormone insulin. Diabetes occurs when either the pancreas doesn’t make enough insulin or when cells of the body don’t respond properly to insulin by taking up glucose.

As a first step to see whether optogenetic regulation of calcium levels in pancreatic cells could trigger insulin release, Toshihiro Kushibiki and colleagues at the National Defense Medical College in Saitama, Japan engineered the channelrhodopsin-1 protein into mouse cells and hit them with laser light of the appropriate frequency. An hour after a short burst of light (a few seconds) the insulin levels had doubled.

The photo below shows a clump of these cells: the nuclei are blue and the channel protein (yellow) can be seen sitting across the cell membranes.

 

Cells expressing a fluorescently tagged channelrhodopsin protein (yellow). Nuclei are blue. From Kushibiki et al., 2015.

 

 

To show that this could work in animals they suspended the engineered cells in a gel and inoculated blobs of the goo under the skin of diabetic mice. Laser burst again: blood glucose levels fell and they showed this was due to the irradiated, implanted cells producing insulin.

Fast forward three years

Those brilliant results highlighted the potential of optogenetic technology as a completely novel approach to a disease that afflicts over 300 million people worldwide.

Scheme showing a Smartphone can be used to regulate the release of insulin from engineered cells implanted in a mouse with diabetes. The key events in the cell are that the light-activated receptor turns on an enzyme (BphS) that in turn controls a transcription regulator (FRTA) that binds to a DNA construct to switch on the Gene Of Interest (GOI) — in this case encoding insulin. (shGLP1, short human glucagon-like peptide 1, is a hormone that has the opposite effect to insulin). From Shao et al., 2017.

In a remarkable confluence of technologies Jiawei Shao and colleagues from a number of institutes in Shanghai, including the Shanghai Academy of Spaceflight Technology, and from ETH Zürich have recently published work that takes the application of optogenetics well and truly into the twenty-first century.

They figured that, as these days nearly everyone lives with their smartphone, the world could use a diabetes app. Essentially they designed a home server SmartController to process wireless signals so that a smartphone could control insulin production by cells in gel capsules implanted in mice. There are differences in the genetic engineering of these cells from those used by Kushibiki’s group but the critical point is unchanged: laser light stimulates insulin release. The capsules carry wirelessly powered LEDs.

The only other thing needed is to know glucose levels. Because mice are only little and they’ve already got their gel capsule, rather than implanting a monitor they took a drop of blood from the tail and used a glucometer. However, looking ahead to human applications, continuous glucose monitors are now available that, placed under the skin, can transmit a radio signal to the controller and, ultimately, it will be possible for the gel capsules to have a built-in battery plus glucose sensor and the whole thing could work automatically.

Any chance of illuminating cancer?

This science is so breathtaking it seems cheeky to ask but, well, I’d say ‘yes but not just yet.’ So long as the ‘drug’ you wish to use can be made biologically (i.e. from DNA by the machinery of the cell), rather than by chemical synthesis, Shao’s Smartphone set-up can readily be adapted to deliver anti-cancer drugs. This might be hugely preferable to the procedures currently in use and would offer an additional advantage by administering drugs in short bursts of lower concentration — a regimen that in some mouse cancer models at least is more effective.

References

Dawydow, A., Kittel, R.J. et al., 2014. Channelrhodopsin-2–XXL, a powerful optogenetic tool for low-light applications. PNAS 111, 13972-13977.

Kushibiki et al., (2015). Optogenetic control of insulin secretion by pancreatic beta-cells in vitro and in vivo. Gene Therapy 22, 553-559.

Shao, J. et al., 2017. Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice. Science Translational Medicine 9, Issue 387, eaal2298.

Through the Smokescreen

For many years I was lucky enough to teach in a cancer biology course for third year natural science and medical students. Quite a few of those guys would already be eyeing up research careers and, within just a few months, some might be working on the very topics that came up in lectures. Nothing went down better, therefore, than talking about a nifty new method that had given easy-to-grasp results clearly of direct relevance to cancer.

Three cheers then for Mikhail Denissenko and friends who in 1996 published the first absolutely unequivocal evidence that a chemical in cigarette smoke could directly damage a bit of DNA that provides a major protection against cancer. The compound bound directly to several guanines in the DNA sequence that encodes P53 – the protein often called ‘the guardian of the genome’ – causing mutations. A pity poor old Fritz Lickint wasn’t around for a celebratory drink – it was he, back in the 1930s, that first spotted the link between smoking and lung cancer.

This was absolutely brilliant for showing how proteins switched on genes – and how that switch could be perturbed by mutations – because, just a couple of years earlier, Yunje Cho’s group at the Memorial Sloan-Kettering Cancer Center in New York had made crystals of P53 stuck to DNA and used X-rays to reveal the structure. This showed that six sites (amino acids) in the centre of the P53 protein poked like fingers into the groove of double-stranded DNA.

x-ray-picCentral core of P53 (grey ribbon) binding to the groove in double-stranded DNA (blue). The six amino acids (residues) most commonly mutated in p53 are shown in yellow (from Cho et al., 1994).

So that was how P53 ‘talked’ to DNA to control the expression of specific genes. What could be better then, in a talk on how DNA damage can lead to cancer, than the story of a specific chemical doing nasty things to a gene that encodes perhaps the most revered of anti-cancer proteins?

The only thing baffling the students must have been the tobacco companies insisting, as they continued to do for years, that smoking was good for you.

And twenty-something years on …?

Well, it’s taken a couple of revolutions (scientific, of course!) but in that time we’ve advanced to being able to sequence genomes at a fantastic speed for next to nothing in terms of cost. In that period too more and more data have accumulated showing the pervasive influence of the weed. In particular that not only does it cause cancer in tissues directly exposed to cigarette smoke (lung, oesophagus, larynx, mouth and throat) but it also promotes cancers in places that never see inhaled smoke: kidney, bladder, liver, pancreas, stomach, cervix, colon, rectum and white blood cells (acute myeloid leukemia). However, up until now we’ve had very little idea of what, if anything, these effects have in common in terms of molecular damage.

Applying the power of modern sequencing, Ludmil Alexandrov of the Los Alamos National Lab, along with the Wellcome Trust Sanger Institute’s Michael Stratton and their colleagues have pieced together whole-genome sequences and exome sequences (those are just the DNA that encode proteins – about 1% of the total) of over 5,000 tumours. These covered 17 smoking-associated forms of cancer and permitted comparison of tobacco smokers with never-smokers.

Let’s hear it for consistent science!

The most obvious question then is do the latest results confirm the efforts of Denissenko & Co., now some 20 years old? The latest work found that smoking could increase the mutation load in the form of multiple, distinct ‘mutational signatures’, each contributing to different extents in different cancers. And indeed in lung and larynx tumours they found the guanine-to-thymine base-pair change that Denissenko et al had observed as the result of a specific chemical attaching to DNA.

For lung cancer they concluded that, all told, about 150 mutations accumulate in a given lung cell as a result of smoking a pack of cigarettes a day for a year.

Turning to tissues that are not directly exposed to smoke, things are a bit less clear. In liver and kidney cancers smokers have a bigger load of mutations than non-smokers (as in the lung). However, and somewhat surprisingly, in other smoking-associated cancer types there were no clear differences. And even odder, there was no difference in the methylation of DNA between smokers and non-smokers – that’s the chemical tags that can be added to DNA to tune the process of transforming the genetic code into proteins. Which was strange because we know that such ‘epigenetic’ changes can occur in response to external factors, e.g., diet.

What’s going on?

Not clear beyond the clear fact that tissues directly exposed to smoke accumulate cancer-driving mutations – and the longer the exposure the bigger the burden. For tissues that don’t see smoke its effect must be indirect. A possible way for this to happen would be for smoke to cause mild inflammation that in turn causes chemical signals to be released into the circulation that in turn affect how efficiently cells repair damage to their DNA.

raleighs_first_pipe_in_england-jpeg

Sir Walt showing off on his return                         to England

Whose fault it is anyway?

So tobacco-promoted cancers still retain some of their molecular mystery as well as presenting an appalling and globally growing problem. These days a popular pastime is to find someone else to blame for anything and everything – and in the case of smoking we all know who the front-runner is. But although Sir Walter Raleigh brought tobacco to Europe (in 1578), it had clearly been in use by American natives long before he turned up and, going in the opposite direction (à la Marco Polo), the Chinese had been at it since at least the early 1500s. To its credit, China had an anti-smoking movement by 1639, during the Ming Dynasty. One of their Emperors decreed that tobacco addicts be executed and the Qing Emperor Kangxi went a step further by beheading anyone who even possessed tobacco.

And paying the price

And paying the price

If you’re thinking maybe we should get a touch more Draconian in our anti-smoking measures, it’s worth pointing out that the Chinese model hasn’t worked out too well so far. China’s currently heading for three million cancer deaths annually. About 400,000 of these are from lung cancer and the smoking trends mean this figure will be 700,000 annual deaths by 2020. The global cancer map is a great way to keep up with the stats of both lung cancer and the rest – though it’s not for those of a nervous disposition!

References

Denissenko, M.F. et al. ( (1996). Preferential Formation of Benzo[a]pyrene Adducts at Lung Cancer Mutational Hotspots in P53.Science 274, 430–432.

Cho, Y. et al. (1994). Crystal Structure of a p53 Tumor Suppressor-DNA Complex: Understanding Tumorigenic Mutations. Science, 265, 346-355.

Alexandrov, L.D. et al. (2016). Mutational signatures associated with tobacco smoking in human cancer. Science 354, 618-622.

Obesity and Cancer

Science, you could say, comes in two sorts. There’s the stuff we more or less understand – and there’s the rest. We’re pretty secure with the earth being round and orbiting the sun, the heart being a pump connected to a network of tubes that keeps us alive, DNA carrying the genetic code – and a few other things. But human beings are curious souls and we tend to be fascinated by what we don’t know and can’t see – why the Dance of the Seven Veils caught on, I guess.

Scientists are, of course, the extreme example – they spend their lives pursuing the unknown (and, as Fred Hoyle gloomily remarked, they’re always wrong and yet they always go on). But in this media era they pay a public price for their doggedness because they get asked the pressing questions of the moment. Is global warning going to finish us off soon, why is British sport generally so poor and – today’s teaser – does being fat make you more likely to get cancer?

A few facts go a long way

The major cancers have become familiar because the numbers afflicted are so staggering – but the one good thing is that the epidemiology can tell us something about the disease. Thus for cancers of the bowel, endometrium, kidney, oesophagus and pancreas and also for postmenopausal breast cancer there is clear evidence that being overweight or obese makes you more susceptible. In other words, if you compare large groups with those cancers to equally large numbers without, the disease groups contain significantly more people who are fat. We should add that the above list is conservative. A number of other cancers are almost certainly more common in those who are overweight (brain, thyroid, liver, ovary, prostate and stomach tumours as well as multiple myeloma, leukaemia, non-Hodgkin lymphoma and malignant melanoma in men).

Sizing up the problem

The usual measure is Body Mass Index (BMI) – your weight (in kilograms) divided by the square of your height (in metres). A BMI of 25 to 29.9 and you’re overweight; over 30 is obese. In England in 2009 just over 61% of adults and 28% of children (aged 2-10) were overweight or obese and of these, 23% of adults and 14% of children were obese. And every year these figures get bigger.

How big is the risk?

Impossible to say exactly – for one thing we don’t know how long you need to be exposed to the risk (i.e. being overweight) for cancer to develop but in 2010 just over 5% of the total of new cancer cases in the UK was due to excess weight. That’s another conservative estimate, but it means at least 17,000 out of 309,000 cases, with bowel and breast cancers being the major sites.

What’s going on?

Showing an association is a good start but the important thing is to find out which molecules make that link. For obesity and cancer detail remains obscure but broad outlines are emerging, summarised in the sketch. In obesity fat (adipose) cells increase in both number and size (so it’s a double problem: more cells – and the fat cells themselves are fatter). As this happens other cells are recruited to adipose tissue and, from this cellular cooperative, signalling proteins are released that have the potential to drive tumours. This picture is similar to that of the microenvironment of tumours themselves, where many types of cell infiltrate the new growth. Initially this inflammatory and immune response aims to kill the tumour but if it fails the balance of signalling shifts so that it actually helps the tumour grow. In addition to signals from fat cells themselves, obesity is usually associated with increased levels of circulating growth hormones (e.g., insulin) and of lipids, both of which may also promote tumour development.

Thus many signals with cancerous potential arise in obese individuals. In principle these could initiate tumour growth or they could accelerate it in cancers that have started to develop independently of obesity. So it is complicated – but at least as new signalling strands emerge they offer new targets for drug therapy.

In obesity abnormal signals from fatty tissue can combine with others arising from perturbed metabolism to help cancers develop

Reference

World Cancer Research Fund (WCRF) Panel on Food, Nutrition, Physical Activity, and the Prevention of Cancer (WCRF, 2007).