Blocking the Unblockable

 

It’s very nearly 40 years since the first human ‘cancer gene’ was identified — in 1982 to be precise. By ‘cancer gene’ we mean a region of DNA that encodes a protein that has a role in normal cell behaviour but that has acquired a mutation of some sort that confers abnormal activity on the protein.

The discovery of RAS ‘oncogene’ activation by DNA and protein mutation stimulated intense activity in unveiling the origins of cancer at the molecular level that has continued to this day. It’s been an exciting and sobering story and RAS has emerged as perhaps the best example you could have of the paradox of cancer. On the one hand it seems startlingly simple: on the other it’s been impenetrably complex.

The simple bit first

Relatively quickly it was shown that there were three closely related RAS genes (KRAS, HRAS & NRAS): they all encode a small protein of just 189 amino acids and they all act as a molecular switches. That means RAS proteins can bind to a small regulator molecule (it’s GTP (guanosine triphosphate) — one of the nucleotides found in DNA and RNA). When that happens RAS changes shape so that it can interact with (i.e. stick to) a variety of effector proteins within the cell. These trigger signalling cascades that ultimately control the activity of genes in the nucleus that control cell proliferation, cell cycle progression and apoptosis (cell death). The switch is flicked off when GTP is converted to GDP — so RAS looses its effector binding capacity.

The other simple bit is that RAS turned out to be one end of the spectrum of DNA damage that can activate an oncogene: the smallest possible change in DNA — mutation of just one base changed one amino acid in the RAS protein and hence its shape. Result: permanently switched on RAS: it’s always stuck to GTP.

Cell signalling. Cells receive many signals from messengers that attach to receptor proteins spanning the outer membrane. Activated receptors turn on relays of proteins. RAS proteins are key nodes that transmit multiple signals. The coloured blocks represent a RAS controlled pathway (a relay of proteins, A, B, C, D) that ‘talk’ to the nucleus, switching on genes that drive proliferation. The arrows diverging from RAS indicate that it regulates many pathways controlling such processes as actin cytoskeletal integrity, cell proliferation, cell differentiation, cell adhesion, apoptosis and cell migration.

Oncogenic RAS and human cancers

We’ve noted that RAS signalling controls functions critical in cancer development and it’s therefore not surprising that it’s mutated, on average, in 22% of all human tumours with pancreatic cancer being an extreme example where 90% of tumours have RAS mutations (the form of RAS is actually KRAS). Those facts, together with the seeming simplicity of its molecular action, put RAS at the top of the target table for chemists seeking cancer therapies. We’ve tried to keep up with events in Mission Impossible, Molecular Dominoes and Where’s that tumour? but the repeated story has been that the upshot of the expenditure of much cash, inspiration and perspiration has, until fairly recently, been zippo. Lots of runners but none that made it into clinical trials. However, that has slowly begun to change over the last ten years and now at least five KRAS-modulating agents are in clinical trials.

A few months back Kevin Lou, Kevan Shokat and colleagues at the University of California published a study of a small molecule, ARS-1620, showing that it inhibited mutant KRAS in lung and pancreatic cancer cells. They screened for other interactions that contribute to the KRAS-driven tumour state and identified two sets of such effectors, one enhancing the engagement of ARS-1620 with its target and others that regulated tumour survival pathways in cells and in vivo. Targetting these synergised with ARS-1620 in suppressing tumour growth.

The RAS switch. Scheme of normal RAS action (top): replacement of a bound guanosine diphosphate (GDP) molecule with guanosine triphosphate (GTP) flips the switch so that RAS can interact with other proteins to turn on downstream signalling pathways that control cell growth and differentiation. Oncogenic RAS (with a single amino acid change at position 12 (Glycine to Valine) blocks the breakdown of bound GTP so the switch is always ‘on’. The new small molecule inhibitor characterized by Canon et al., AMG 510, interacts with KRASG12C to block GTP binding. The switch remains ‘off’ and the cancer-promoting activity of mutant KRAS is inhibited.

More recently Jude Canon at Amgen Research, together with colleagues from a number of institutes, described another small molecule, AMG 510, that also recognises the mutant form of KRAS with high specificity, hence impairing cell proliferation. In mice carrying human pancreatic tumours AMG 510 caused permanent tumour regression — provided the mice had functioning immune systems. In mice lacking T cells (i.e. ‘nude’ mice) the tumours re-grew but combining AMG 510 with immunotherapy (an antibody against anti-PD1) gave complete tumour regression. AMG 510 stimulated the expression of inflammatory chemokines that promoted infiltration of the tumours by T cells and dendritic cells (sometimes called ‘antigen-presenting cells’, these cells process antigens and present fragments thereof on their surface to T cells and B cells to promote the adaptive immune response). In preliminary trials four patients with non-small cell lung cancer showed significant effects — either tumour shrinkage or complete inhibition of growth.

So maybe at long last the enigma of RAS is being prised open. The efficacy of AMG 510 against lung cancers is particularly heartening as there remains little in the way of therapeutic options for these tumours.

References

Canon, J. et al. (2019). The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 575, 217–223.

Lou, K. et al. (2019). KRASG12C inhibition produces a driver-limited state revealing collateral dependencies. Science Signaling 12, Issue 583, eaaw9450. DOI: 10.1126/scisignal.aaw9450

Non-Container Ships

 

A question often asked about cancer is: “Can you catch it from someone else?” Answer: “No you can’t.” But as so often in cancer the true picture requires a more detailed response — something that may make scientists unpopular but it’s not our fault! As Einstein more or less said “make it as simple as possible but no simpler.”

No … but …

So we have to note that some human cancers arise from infection — most notably by human immunodeficiency viruses (HIV) that can cause acquired immunodeficiency syndrome (AIDS) and lead to cancer and by human papillomavirus infection (HPV) that can give rise to lesions that are the precursors of cervical cancer. But in these human cases it is a causative agent (i.e. virus) that is transmitted, not tumour cells.

However, there are three known examples in mammals of transmissible cancers in which tumour cells are spread between individuals: the facial tumours that afflict Tasmanian devils, a venereal tumour in dogs and a sarcoma in Syrian hamsters.

Not to be outdone, the invertebrates have recently joined this select club and we caught up with this extraordinary story in Cockles and Mussels, Alive, Alive-O! It’s a tale of clams and mussels and various other members of the huge family of bivalve molluscs — (over 15,000 species) — that began 50 years ago when some, living along the east and west coasts of North America and the west coast of Ireland, started to die in large numbers. It turned out that the cause was a type of cancer in which some blood cells reproduce in an uncontrolled way. It’s a form of leukemia: the blood turns milky and the animals die, in effect, from asphyxiation. In soft-shell clams the disease had spread over 1,500 km from Chesapeake Bay to Prince Edward Island but the really staggering fact came from applying the power of DNA sequencing to these little beach dwellers. Like all cancers the cause was genetic damage — in this case the insertion of a chunk of extra DNA into the clam genome. But amazingly this event had only happened once: the cancer had spread from a single ‘founder’ clam throughout the population. The resemblance to the way the cancer spreads in Tasmanian devils is striking.

Join the club

In 2016 four more examples of transmissible cancer in bivalves were discovered — in mussels from British Columbia, in golden carpet shell clams from the Spanish coast and in two forms in cockles. As with the soft-shell clams, DNA analysis showed that the disease had been transmitted by living cancer cells, descended from a single common ancestor, passing directly from one animal to another. In a truly remarkable twist it emerged that cancer cells in golden carpet shell clams come from a different species — the pullet shell clam — a species that, by and large, doesn’t get cancer. So they seem to have come up with a way of resisting a cancer that arose in them, whilst at the same time being able to pass live tumour cells on to another species!!

Map of the spread of cancer in mussels. This afflicts the Mytilus group of bivalve molluscs (i.e. they have a shell of two, hinged parts). BTN = bivalve transmissible neoplasias (i.e. cancers). BTN 1 & BTN2 indicates that two separate genetics events have occurred, each causing a similar leukemia. The species involved are Mytilus trossulus (the bay mussel), Mytilus chilensis (the Chilean blue mussel) and Mytilus edulis (the edible blue mussel). The map shows how cancer cells have spread from Northern to Southern Hemispheres and across the Atlantic Ocean. From Yonemitsu et al. (2019).

Going global

In the latest instalment Marisa Yonemitsu, Michael Metzger and colleagues have looked at two other species of mussel, one found in South America, the other in Europe. DNA analysis showed that the cancers in the South American and European mussels were almost genetically identical and that they came from a single, Northern hemisphere trossulus mussel. However, this cancer lineage is different from the one previously identified in mussels on the southern coast of British Columbia.

Unhappy holidays

It seems very likely that some of these gastronomic delights have hitched a ride on vessels plying the high seas so that carriers of the cancer have travelled the oceans. Whilst one would not wish to deny them the chance of a holiday, this is serious news because of the commercial value of seafood.

It’s another example of how mankind’s advances, in this case being able to build things like container ships with attractive bottoms, for molluscs at least, can lead to unforeseen problems.

This really bizarre story has only come light because of the depletion of populations of clams and mussels in certain areas but it certainly carries the implication that transmissible cancers may be relatively common in marine invertebrates.

Reference

Yonemitsu, M.A. et al. (2019). A single clonal lineage of transmissible cancer identified in two marine mussel species in South America and Europe. eLife 2019;8:e47788 DOI: 10.7554/eLife.47788.

Brainstorming

 

It’s the first day of a New Year and, as is well known, Scottish folk world-wide make a big celebration of yesterday (Hogmanay), New Year’s Day and indeed quite often the next few days for good measure. Even in the far north-west of England as a youngster with more or less black hair (deemed to be important for some reason) I was trundled round the neighbours in one of the rituals — ‘first-footing’, i.e. being the first guest of the new year, despite our family having no Scottish connections that I knew of.

Scots Wha Hae

Most such jollifications seem to require mournful dirges accompanying incomprehensible lyrics by Robert Burns. To be fair I should note that Max Bruch and Hector Berlioz, wonderful composers both, saw fit to include a musical reference to ‘Scots Wha Hae’ in the Scottish Fantasy and in the concert overture Rob Roy. Mind you, Berlioz himself described his overture as “long and diffuse” and it was so badly received that he burned the score the night of its premier.

However, there is something else that Scots make quite a fuss about, given half a chance, and here perhaps we can agree they have a point. It’s the number of notable scientists and physicians their country has produced. Wikipedia’s List of Scottish engineers and scientists runs to over 150 names — remarkable for a population that even today is only about five million. The listed luminaries feature some household names: Alexander Graham Bell, James Watt, James Clerk Maxwell, Lord Kelvin and Joseph Lister just to be going on with.

But there’s a slightly unnerving thing about Wikipedia’s List in that, long though it is, there are some serious omissions. I spotted this the other day when I was searching for a bit of background about one of the heroes of this New Year’s story. The first missing star I noted was John Hunter, generally thought to have carried out the first surgical removal of a malignant melanoma (skin cancer) in 1787. Worse still, I found no mention of William Macewen: it was his first successful removal of a brain tumour (in 1879) that makes him directly relevant to our story. He was a truly remarkable figure. Thought of as the ‘Father of neurosurgery’, he was a pioneer in  surgery of the brain and other organs. But the really outstanding thing about Sir William Macewen CB., FRS., FRCS, to give him his full handle, was his approach to surgery. Thus, for example, in treating brain tumours he applied his profound knowledge of anatomy to work out from the patient’s symptoms the precise location of the abnormal growth so he knew where to take surgical aim. Amazing!

Very slow progress

Nearly 60 years after Macewen’s pioneering surgery the American composer George Gershwin would have appreciated his genius as treatments had made little progress by the 1930s when Gershwin succumbed to a brain tumour (specifically a glioblastoma multiforme). It took until 1958 for the first useful drug treatment for brain tumours to emerge and until the mid-1970s for radiation therapy come into use. Indeed it was only the introduction of CT scans towards the end of the 20th century that permitted tumour localisation without needing Macewen’s extraordinary gifts.

Something very odd

In parallel with these advances has emerged the evidence for an unexpected feature of brain tumours. You might guess that brain tumours would start in the brain but it turns out that most do nothing of the sort. The vast majority (about 90%) are secondary cancers: that is, they arise when tumour cells spread from another part of the body — commonly breast or lung. In other words most brain tumours are metastases — and they are mighty important. About 24,000 people in the United States discover they have these abnormal growths every year and they cause about 18,000 deaths. The rates are much the same in the UK where deaths from brain and related tumours number just over 5,000.

But also familiar …

Those who follow developments on cancer will know that metastasis is one of the hottest potatoes. Until very recently we had no idea of the molecular goings on that turn a cell in a primary tumour into a wanderer that can leave its site of origin, get into the bloodstream, get out at some other location and there establish a new, secondary colony. The mists are beginning to lift as the wonders of modern biology are applied to this pressing problem.

Step forward one of the main movers and shakers in the field who is the modern hero of today’s piece: David Lyden of the Gale and Ira Drukier Institute for Children’s Health, Weill Cornell Medicine, New York.

So topical is this issue of metastasis that I’m relieved to note that the contributions of the Lyden group have featured regularly in these pages (Keeping Cancer CatatonicScattering the Bad Seed and Holiday Reading (4) – Can We Make Resistance Futile). A succinct summary of those contributions would be: (1) cells in primary tumours release ‘messengers’ into the circulation that ‘tag’ metastatic sites before any cells actually leave the tumour, (2) the messengers that do the site-tagging are small sacs — mini cells — called exosomes, and (3) they find specific addresses by carrying protein labels that home in to different organs — we represented that in the form of a tube train map in Lethal ZIP Codes.

In One More Small Step the same team looked closely at exosomes and found that a wide variety of tumour cell types secrete two sizes of exosomes (big and small! — see blog for details!!). Amazingly these sacs carry about 1000 different types of protein — suggesting that they might have powerful effects.

Breaking the barrier

With that in mind Lyden’s group have now turned their attention to how tumour cells find their way to the brain. How do they achieve the feat of crossing the ‘blood-brain barrier’ — the layer of (endothelial) cells that encloses the brain and controls the types of molecules that can move to and from circulating blood — and are exosomes involved? In other words, are they little bags of trouble that play a role in helping most brain tumours to grow?

Answer ‘yes’ of course, or we wouldn’t have spent so long getting up to speed on the subject. Gonçalo Rodrigues, Lyden & Co. set up a brain slice culture system and pre-treated the slices with exosomes from human breast cancer metastatic cells that were known to spread preferentially to different tissues (brain, lung or bone).

Photos of brain slices showing how exosomes help to provide a niche for human breast cancer metastatic cells to invade, attach and grow. These are fluorescence microscopy images: brain blood vessels (vasculature) are red; cancer cells are green (GFP). Left: no pre-treatment; Right: pretreatment with exosomes. White arrowheads show vasculature-associated cancer cells. White bar = 100 micronsFrom Rodrigues et al. 2019.

The photos show a typical experiment using brain-seeking exosomes. There is a huge increase in the number of green cancer cells attaching to the brain slice as a result of exosome pre-treatment (right) by comparison with no exosome addition (left). Corresponding experiments with exosomes that direct migration to lung or bone show no effect: cancer cell attachment remains low (as in the left hand photo).

How do they do it?

The group took their studies a stage further by looking at the 1000 or so proteins in the exosomes for any that seemed to specify migration to the brain — in other words, to act as addresses of the kind we described in Lethal ZIP Codes. They came up with one in particular: a protein called CEMIP  (if you’re interested that stands for ‘cell migration inducing hyaluronidase 1’. It’s an enzyme that chops up long chains of sugars (called hyaluronic acid). These chains form scaffolds to support proteins in various tissues including the brain — and their disruption may play a role in cancer cell movement).

The levels of CEMIP are higher in exosomes that promote brain metastasis but not in those associated with lung or bone metastatic cells. Thus pre-conditioning the brain microenvironment with CEMIP+ exosomes drives invasion. When they are depleted invasion and tumour cell association with the brain vasculature is disrupted. This remarkable new work has revealed how exosomes help wandering tumour cells to storm the blood-brain barrier. Immediately this opens the possibility of isolating exosomes from small samples of blood and screening them for proteins — i.e. using them as a ‘biomarker’ for metastatic cancer targets. But of course the great goal is to be able to interfere with their actions, an intervention that could dramatically cut the incidence of brain tumours. What a triumph that would be!!

We began with a Scottish tradition. Let’s end with another by raising a mental glass to scientists all over the world who, step by perspiring step are inching towards the goal of controlling cancer. Keep it up guys — and back to your benches!!

Reference

Rodrigues et al. (2019). Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis. Nature Cell Biology 21, 1403–1412.

 

 

Little Things That May Mean a Lot

 

You may have noticed a seeming oddity about science in that you often hear nothing about a topic for ages and then along come several new pieces of work more or less together — the London bus effect. There’s number of reasons for this, one being that scientists love gadgets — they’re really little boys and girls with licence to play with their toys for a living — so when a new method or piece of kit appears there’s usually something of a band wagon response. Another factor is that different labs quite often talk to each other and this can lead to collaborative efforts sometimes resulting in several, complementary publications. We’ve seen this recently with bugs and their effect on human cancers. In Secret Army: More Manoeuvres Revealed we saw how bacteria could drive lung cancer and in Mushrooming Secret Army how fungi are now established as players in at least in one type of cancer.

Now add to these a paper by Hila Sberro, Ami Bhatt and colleagues from Stanford, Berkeley and the Biomedical Sciences Research Center Alexander Fleming, Vari, Greece that reveals a huge pool of hitherto unknown proteins in the human microbiome.

What Sberro & Co did was to take tissue samples (1,773 of them) from humans (skin, vagina, gut and mouth) and look at the DNA sequences therein. What you get doing this is the ‘metagenome’ — i.e., the DNA of the whole community you pick up — and that type of study is therefore called ‘comparative genomics’.

Scheme showing how metagenomic analysis can identify thousands of small coding regions of DNA from microbiome sequences obtained from a range of human tissues. From Sberro et al., 2019.

They focused on ‘small’ proteins of 50 or fewer amino acids. The hormone insulin has 51 amino acids and proteins in the size range up to about 50 amino acids are often called ‘peptides’. Perhaps counter-intuitively, large proteins are easier to isolate than the little chaps who have for this reason been rather overlooked — until now that is.

Some over-sight because Sberro et al. discovered more than 400,000 of these potential mini-proteins lurking in the nooks and crannies of their human volunteers. This hitherto largely unknown horde (fewer that 5% had been identified before) turned out to be made up of about 4,500 ‘families’ — groups of proteins that are similar in size and amino acid content.

This is a really astonishing finding quite literally under our noses. At the moment we have no idea what most of these bacterial proteins do. As you might expect, some of the proteins appear to be involved in keeping cells alive (they’re ‘housekeeping genes’). You might also guess that some may not have any role at all — they’re just a kind of accidental by-product — but, by and large, Nature doesn’t waste energy and making proteins is a very expensive business in energetic terms. And if you’re in any doubt about the importance of ‘peptides’, give a moment’s thought to the human proteins oxytocin (9 amino acids that plays an important role in sexual reproduction and in childbirth) and — even smaller — the tripeptide (i.e. 3 amino acids) glutathione that protects most living things from damage by free radicals.

As some of the small, bacterial proteins are present in large amounts we can be confident they too do something useful — perhaps protect the bacteria themselves from their own toxins, made to kill viruses.

And, as ever, when we get to understand what these little guys are up to they may be useful in, for example, interventional medicine.

Reference

Sberro, H. et al., 2019. Large-Scale Analyses of Human Microbiomes Reveal Thousands of Small, Novel Genes. Cell 178, 1-15.

Breaking Up Is Hard To Do

 

Thus Neil Sedaka, the American pop songster back in the 60s. He was crooning about hearts of course but since then we’ve discovered that for our genetic hearts — our DNA — breaking up is not that tough and indeed it’s quite common.

A moving picture worth a thousand words

When I’m trying to explain cancer to non-scientists I often begin by showing a short movie of a cell in the final stages of dividing to form two identical daughter cells. This is the process called mitosis and the end-game is the exciting bit because the cell’s genetic material, its DNA, has been duplicated and the two identical sets of chromosomes are lined up in the middle of the cell. There ensues a mighty tug-of-war as cables (strands of proteins) are attached to the chromosomes to rip them apart, providing a separate genome for each new cell when, shortly after, the parent cell splits into two. When viewed as a speeded-up movie it’s incredibly dramatic and violent — which is why I show it because it’s easy to see how things could go wrong to create broken chromosomes or an unequal division of chromosomes (aneuploidy). It’s the flip side if you like to the single base changes (mutations) — the smallest damage DNA can suffer — that are a common feature of cancers.

In Heir of the Dog we showed pictures of normal and cancerous chromosomes that had been tagged with coloured markers to illustrate the quite staggering extent of “chromosome shuffling” that can occur.

Nothing new there

We’ve known about aneuploidy for a long time. Over 20 years ago Bert Vogelstein and his colleagues showed that the cells in most bowel cancers have different numbers of chromosomes and we know now that chromosomal instability is present in most solid tumours (90%).

Knowing it happens is one thing: being able to track it in real time to see how it happens is another. This difficulty has recently been overcome by Ana C. F. Bolhaqueiro and her colleagues from the Universities of Utrecht and Groningen who took human colorectal tumour cells and grew them in a cell culture system in the laboratory that permits 3D growth — giving rise to clumps of cells called organoids.

Scheme representing how cells grown as a 3D clump (organoid) can be sampled to follow chromosomal changes. Cells were taken from human colon tumours and from adjacent normal tissue and grown in dishes. The cells were labelled with a fluorescent tag to enable individual chromosomes to been seen by microscopy as the cells divided. At time intervals single cells were selected and sequenced to track changes in DNA. From Johnson and McClelland 2019.

Genetic evolution in real time

As the above scheme shows, the idea of organoids is that their cells grow and divide so that at any time you can select a sample and look at what’s happening to its DNA. Furthermore the DNA can be sequenced to pinpoint precisely the genetic changes that have occurred.

It turned out that cancer cells often make mistakes in apportioning DNA between daughter cells whereas such errors are rare in normal, healthy cells.

It should be said that whilst these errors are common in human colon cancers, a subset of these tumours do not show chromosomal instability but rather have a high frequency of small mutations (called microsatellite instability). Another example of how in cancer there’s usually more than one way of getting to the same end.

Building bridges …

The most common type of gross chromosomal abnormality occurs when chromosomes fuse via their sticky ends to give what are called chromatin bridges (chromatin just means DNA complete with all the proteins normally attached to it). Other errors can give rise to a chromosome that’s become isolated — called a lagging chromosome, it’s a bit like a sheep that has wandered off from the rest of the flock. As the cell finally divides and the daughter cells move apart, DNA bridges undergo random fragmentation.

… but where to …

Little is known about how cells deal with aneuploidy and the extent to which it drives tumour development. This study showed that variation in chromosome number depends on the rate at which chromosomal instability develops and the capacity of a cell to survive in the face of changes in chromosome number. More generally for the future, it shows that the organoid approach offers an intriguing opening for exploring this facet of cancer.

Reference

Bolhaqueiro, A.C.F. et al. (2019). Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids. Nature Genetics 51, 824–834.

Lengauer, C. et al., (1997). Genetic instability in colorectal cancers. Nature 386, 623-627.

Johnson, S.C. and McClelland, S.E. (2019). Watching cancer cells evolve. Nature 570, 166-167.

Sticky Cancer Genes

 

In the previous blog I talked about Breath Biopsy — a new method that aims to detect cancers from breath samples. I noted that it could end up complementing liquid biopsies — samples of tumour cell DNA pulled out of a teaspoon of blood — both being, as near as makes no difference, non-invasive tests. Just to show that there’s no limit to the ingenuity of scientists, yet another approach to the detection problem has just been published — this from Matt Trau and his wonderful team at The University of Queensland.

This new method, like the liquid biopsy, detects DNA but, rather than the sequence of bases, it identifies an epigenetic profile — that is, the pattern of chemical tags (methyl groups) attached to bases. As we noted in Cancer GPS? cancer cells often have abnormal DNA methylation patterns — excess methylation (hypermethylation) in some regions, reduced methylation in others. Methylation acts as a kind of ‘fine tuner’, regulating whether genes are switched on or off. In the methylation landscape of cancer cells there is an overall loss of methylation but there’s an increase in regions that regulate the expression of critical genes. This shows up as clusters of methylated cytosine bases.

Rather helpfully, a little while ago in Desperately SEEKing … we talked about epigenetics and included a scheme showing how differences in methylation clusters can identify normal cells and a variety of cancers and how these were analysed in the computer program CancerLocator.

The Trau paper has an even better scheme showing how the patterns of DNA decoration differ between normal and cancer cells and how this ‘methylscape’ (methylation landscape) affects the physical behaviour of DNA.

How epigenetic changes affect DNA. Scheme shows methylation (left: addition of a methyl group to a cytosine base in DNA) in the process of epigenetic reprogramming in cancer cells. This change in the methylation landscape affects the solubility of DNA and its adsorption by gold (from Sina et al. 2018).

Critically, normal and cancer epigenomes differ in stickiness — affinity — for metal surfaces, in particular for gold. In a clever ploy this work incorporated a colour change as indicator. We don’t need to bother with the details — and the result is easy to describe. DNA, extracted from a small blood sample, is added to water containing tiny gold nanoparticles. The colour indicator makes the water pink. If the DNA is from cancer cells the water retains its original colour. If it’s normal DNA from healthy cells the different binding properties turns the water blue.

By this test the Brisbane group have been able to identify DNA from breast, prostate and colorectal cancers as well as from lymphomas.

So effective is this method that it can detect circulating free DNA from tumour cells within 10 minutes of taking a blood sample.

The aim of the game — and the reason why so much effort is being expended — is to detect cancers much earlier than current methods (mammography, etc.) can manage. The idea is that if we can do this not weeks or months but perhaps years earlier, at that stage cancers may be much more susceptible to drug treatments. Trau’s paper notes that their test is sensitive enough to detect very low levels of cancer DNA — not the same thing as early detection but suggestive none the less.

So there are now at least three non-invasive tests for cancer: liquid biopsy, Breath Biopsy and the Queensland group’s Methylscape, and in the context of epigenetics we should also bear in mind the CancerLocator analysis programme.

Matt Trau acknowledges, speaking of Methylscape, that “We certainly don’t know yet whether it’s the holy grail for all cancer diagnostics, but it looks really interesting as an incredibly simple universal marker for cancer …” We know already that liquid biopsies can give useful information about patient response to treatment but it will be a while before we can determine how far back any of these methods can push the detection frontier. In the meantime it would be surprising if these tests were not being applied to age-grouped sets of normal individuals — because one would expect the frequency of cancer indication to be lower in younger people.

From a scientific point of view it would be exciting if a significant proportion of ‘positives’ was detected in, say, 20 to 30 year olds. Such a result would, however, raise some very tricky questions in terms of what, at the moment, should be done with those findings.

Reference

Abu Ali Ibn Sina, Laura G. Carrascosa, Ziyu Liang, Yadveer S. Grewal, Andri Wardiana, Muhammad J. A. Shiddiky, Robert A. Gardiner, Hemamali Samaratunga, Maher K. Gandhi, Rodney J. Scott, Darren Korbie & Matt Trau (2018). Epigenetically reprogrammed methylation landscape drives the DNA self-assembly and serves as a universal cancer biomarker. Nature Communications 9, Article number: 4915.

Shifting the Genetic Furniture

 

Readers of these pages will know very well that cells are packets of magic. Of course, we often describe them in the simplest terms: ‘Sacs of gooey stuff with lots of molecules floating around.’ And it’s true that we know a lot about the protein pathways that capture energy from the food we eat and about the machinery that duplicates genetic material, makes new proteins and sustains life. Even so, although we’ve worked out much molecular detail, we have scarcely a clue about how ‘stuff’ in cells is organised. How do the tens of thousands of different types of proteins find their places in the seemingly chaotic jumble of a cell so that they can do their job? If that remains a mystery there’s an even more perplexing one in the form of the nucleus. That’s a smaller sac (i.e. a compartment surrounded by a membrane) that is home to most of our genetic material — i.e. DNA.

Sizing up the problem

It’s easy to see why evolution came up with the idea of a separate enclosure for DNA which only has to do two things: reproduce itself and enable regions of its four base code to be transcribed into molecules that can cross the nuclear membrane to be translated into proteins in the body of the cell. But here’s the puzzle. The nucleus is very small and there’s an awful lot of DNA — over 3,000 million bases in each of the two strands of human DNA (and, of course, two complete sets of chromosomes go to make up the human genome) — so 2 metres of it in every cell. A rather pointless exercise, unless you go in for pub quizzes, is to work out the length of all your DNA if you put it together in a single string. 1013 cells (i.e. 1 followed by 13 zeros) in your body: 2 metres per cell. Answer: your DNA would stretch to the sun and back 67 times.

Mmm. More relevantly, the nucleus of a cell is typically about 6 micrometres (µm) in diameter — that’s six millionths of a metre (6/1,000,000 metre), into which our 2 metres must squeeze.

Time for some serious packing to be done but it’s not just a matter of stuffing it in any old how and sitting on the lid. As we’ve just noted, every time cells divide all the DNA has to be replicated and regions (i.e. genes) are continually being “read” to make proteins. So the machinery in the nucleus has to be able to get at specific regions of DNA and disentangle them sufficiently for code reading. Part of evolution’s solution to these problems has been to add proteins called histones to DNA (the term chromosome refers to DNA together with histone packaging proteins and other proteins). To understand how this leads to “more being less”, consider DNA as a length of cotton. If you just scrunch the cotton up into a ball you get a tangled mess. But if you use cotton reels (aka histones — two or three hundred million per cell), you can reduce the great length to smaller, more organized blocks — which is just as well because they’re all that stands between life and a tangled mess.

Thinking of histones as cotton reels helps a bit in thinking about how the nucleus achieves the seemingly impossible but the fact of the matter is that we have no real idea about how DNA is unravelling is controlled so that the two strands can be unzipped and replicated, yet alone the way in which starting points for reading genes are found by proteins.

Undeterred by our profound ignorance Haifeng Wang and colleagues at Stanford University have just done something really amazing. They came up with a way of moving regions of DNA from the jumble of the nuclear interior to the membrane and they showed that this can change the activity of genes. They used CRISPR (that we described in Re-writing the Manual of Life) to insert a short piece of DNA next to a chosen gene. The insert was tagged with a protein designed to attach to a hormone that also binds to a protein (called emerin) that sits in the nuclear membrane. So the idea was that when the hormone is added to cells it can hook on to the DNA tag and, by attaching to emerin, can drag the chosen gene to the membrane. The coupling agent is a plant hormone (abscisic acid) although it also occurs in other species, including humans. Wang & Co christened their method CRISPR-GO for CRISPR-Genome Organizer.

Tagging a DNA insert with a protein so that a coupling molecule can pull a region of DNA to a protein in the membrane of the nucleus. From Wang et al., 2018.

Repositioning regions of DNA in the nucleus. DNA is labeled blue which defines the shape of the nucleus. Red dots are specific genes before (left) and after (right) adding the coupling agent. From Pennisi 2018.

How did CRISPR-GO go?

Astonishingly well. Not only could it shift tagged DNA from the interior to the membrane of the nucleus but the rearrangements could change the way cells behaved. Depending on which regions were moved and where to, cells grew more slowly or more rapidly.

So this is a really remarkable technical feat — but it’s not just molecular pyrotechnics for fun. It looks as though this approach may offer at long last a way of dissecting how cells go about getting a controlled response out of the mind-boggling complexity that is their genetic material.

References

Wang, H. et al. (2018). CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization. Cell 175, 1405-1417.

Pennisi, E. (2018). Moving DNA to a different part of the nucleus can change how it works. Science Oct. 11th.

Taking Aim at Cancer’s Heart

 

Cancer is a unique paradox. At one level it’s as easy as can be to describe: damage to DNA (aka mutations) drives cells to behave abnormally — to make more of themselves when they shouldn’t.

But we all know that cancer’s fiendishly complicated — at least at the level of fine detail. Over the last decade or so the avalanche of sequenced DNA has revealed that every cell in a tumour is different: compare one cell to its neighbour and you’ll find variations in the individual units (the bases A, C, G & T) that make up the chains of DNA.

It’s a nightmare: every cancer is different so we need an infinite number of treatments to control or cure each one. Time to give up and retire to the pub.

Drivers and passengers

Not quite. DNA sequencing has also revealed that, amongst all the genetic mayhem, some mutations are more important than others. The movers and shakers have been dubbed ‘drivers’: those that come along for the ride are ‘passengers’. The hangers-on are heavily in the majority but, even so, several hundred drivers (i.e. mutated genes that give rise to abnormal proteins) have been identified. As it needs a group of drivers to work together to make a cancer we still have the problem that the number of critical combinations that can arise is essentially infinite.

One way of reducing the scale of the problem has been to look at what ‘driver’ proteins do in cells and to target those acting at key points to push cell proliferation beyond the normal.

Playing games

Just recently Giulio CaravagnaAndrea Sottoriva and colleagues at the Institute of Cancer Research, London and the University of Edinburgh have come up with a different approach. The idea goes back to the 1950s when a clever chap from Kansas by the name of Arthur Samuel came up with a program for IBM’s first commercial computer so that it could play draughts (checkers as our American friends call it) in its spare time. The program defined the patterns that could be formed by the pieces on the chequerboard so that, given enough of these, IBM 701 could indicate the optimal moves. Samuel called this machine learning, a precursor of the idea of artificial intelligence.

Perhaps the most famous moment in this saga came in 1997 when a later IBM computer, Deep Blue, beat the then world chess champion Garry Kasparov. Unsurprisingly, Kasparov was a bit miffed and accused IBM of cheating — to wit, getting a human to tell the machine what to do. Let’s hope that in the end he came to terms with the fact that Deep Blue could crank through 200 million positions per second and, however many games Grandmasters have in their heads, they can’t compete with that.

The cancer team realized that the mutations driving the evolution of cancer cells emerge as patterns in the sequence of DNA as a cell moves towards becoming independent of normal controls. Think of each cancer as a family tree of mutations, the key question being which branch leads to the most potent combination.

To pick out these patterns they applied a machine-learning approach known as transfer learning to the DNA sequences from a large number of cancers. They called this ‘repeated evolution in cancer’ — REVOLVER — aimed at picking out mutation patterns at the heart of cancer that foreshadow future genetic changes and can be used to predict how they will evolve.

Identifying patterns of mutation common to different tumours.

Samples are taken from different regions of a patient’s tumour (represented by the coloured dots). Their DNA sequences will have multiple variations that can mask underlying patterns of driver mutations present in some subgroups. The five trees show mutations picked up in those patients. REVOLVER uses transfer learning to screen the sequence data from many patients and pull out evolutionary trajectories shared by subgroups. The dotted red lines highlight common patterns that are represented in the lower strip. From Caravagna et al. 2018.

REVOLVER was applied to sequences from lung, breast, kidney and bowel cancers but there’s no reason it shouldn’t work with other tumours. The big attraction is that if these mini-sequence mutation patterns can be associated generally with how a given tumour develops they should help to inform treatment options and predict survival.

We have in the past referred to the ways cancers evolve as ‘genetic roulette’ — so perhaps it’s appropriate if game-playing computer programs turn out to be useful in teasing out behavioural clues.

Reference

Caravagna, G. et al. (2018). Detecting repeated cancer evolution from multi-region tumor sequencing data. Nature Methods 15, 707–714.

Now wash your hands!

 

You must have spent the last 20 years on a distant planet if you’re unaware that we’re heading for Antibiotic Armaggedon — the rise of “Superbugs”, i.e., bacteria resistant to once-successful medication. Microbes resistant to multiple antimicrobials are called multidrug resistant. It’s a desperate matter because it means trivial infections may become fatal and currently safe surgical procedures may become dangerous.

Time-line of the discovery of different antibiotic classes in clinical use. The key point is that the last antibiotic class to become a successful treatment was discovered in 1987.

What’s the problem?
It’s 30 years since we came up a new class of antibiotics. The golden age launched by Fleming’s celebrated discovery of penicillin is long gone and while the discovery curve has drifted ever downwards since 1960 the bugs have been busy.

Just how busy a bug can be was shown by a large-scale experiment carried out by Roy Kishony and friends. They built a “Mega-Plate” — a Petri Dish 2 ft by 4 ft filled with a jelly for the bacteria to grow in. The bugs were seeded into channels at either end so they would grow towards the middle. The only thing stopping them was four channels dosed with antibiotic at increasing concentrations — 10 times more in each successive channel.

The bugs grow until they hit a wall of antibiotic. There they pause for a think — and, after a bit, an intrepid little group start to make their way into the higher dose of drug. Gradually the number of groups expand until a tidal wave sweeps over that barrier. This is repeated at each new ‘wall’ — four times until the whole tray is a bug fest.

When they pause at each new ‘wall’ they’re not ‘thinking’ of course. They’re just picking up random mutations in their DNA until they are able to advance into the high drug environment. So this experiment is a fantastic visual display of bugs becoming drug-resistant. And it’s terrifying because it takes about 11 days for them to overcome four levels of drug. It’s even more scary in the speeded-up movie as that lasts less than two minutes.

Sound familiar?
It should do as this is a cancer column and readers will know that cancers arise by picking up mutations. To highlight the similarities the picture below is the left-hand half of the bug tray with new colonies shown as linked dots. You could perfectly well think of these as early stage cancer cells acquiring mutations in ‘driver’ genes that push them towards tumour formation.

So that’s pretty scary too and the only good news is that animal cells reproduce much more slowly than bacteria. The fastest they can manage is about 48 hours to grow and divide into two new cells and for many it’s much slower than that. Bugs, on the other hand, can do it in 20 minutes if you feed them enough of the right stuff.

Which is why we don’t all get zonked by cancer at an early age.

The evolution of bacteria on a “Mega-Plate” Petri Dish. The vertical red lines mark the boundaries of increasing antibiotic concentrations. You could equally think of each dot that represents a new bacterial colony being early stage cancer cells acquiring mutations in ‘driver’ genes (white arrows) that push them towards tumour formation. From Roy Kishony’s Laboratory at Harvard Medical School.

Enough of that!
But for once I don’t want to talk about cancer but about a really fascinating piece of work that caught my eye in the journal Cell Reports. It’s by Gianni Panagiotou, Kang Kang and colleagues from The University of Hong Kong and The Hans Knöll Institute, Jena, Germany and it’s all about their travels on the Hong Kong MTR (Mass Transit Railway). This is the network of over 200 km of railway lines with 159 stations that serves the urbanised areas of Hong Kong IslandKowloon, and the New Territories and has a cross- border connection to the neighboring city of Shenzhen in mainland China.

An MTR train on the Tung Chung line that links Lantau Island with Hong Kong Island.

Being scientists of course they weren’t just having a day out. They wanted to know the contents of the microbiome that they and their fellow travellers picked up on the palms of their hands when riding the rails. ‘Microbiome’ means all of the collection of microorganisms — though in practice this is almost entirely bacteria. So they swabbed the palms of volunteers and then threw the full power of modern DNA sequencing and genetic analysis at what they’d scraped off. Or, as they put it: “We conducted a metagenomic study of the Hong Kong MTR system.”

And if you’re thinking it might be possible to take a trip on the Hong Kong Metro without grabbing a handrail or otherwise engaging in what on the London Underground used to be called ‘strap-hanging’ you clearly haven’t tried it!

Hong Kong MTR.

 

The MTR System and Sampling Procedure. Left: The eight urban lines studied: the Airport Express line and Disneyland Resort branch were excluded. The Central-Hong Kong station and the cross-border rail stations connecting with the MTR and the Shenzhen metro system are labeled. Right: The sampling procedure included handwashing, handrail touching for 30 min and swabbing. From Kang et al. 2018.

Hold very tight please! 

It’s going to become a seriously bumpy ride. The major findings were:

  1. Four groups (phyla) of bacteria dominated: Actinobacteria [51%], Proteobacteria [27%], Firmicutes [11%] and Bacteroidetes [2%]. Followers of this blog will be delighted to spot the last two (B & F) as we’ve met them several times before (in Hitchhiker Or Driver?, Fast Food Fix Focuses on Fibre, Our Inner Self, The Best Laid Plans In Mice and Men, and, of course, in it’s a small world) — that’s how important they are in the context of cancer.
  2. The dominant organism (29% of the community) was P. acnes (one of the Actinobacteria — it’s the bug linked to the skin condition of acne).
  3. Some non-human-associated species (e.g., soil organisms) also popped up that varied enormously in amount from day to day — perhaps because of weather conditions (e.g., humidity).
  4. Variation in the make-up of the microbial communities picked up depended, more than anything else, on the time of day. There was a marked decrease in diversity in afternoon samples compared with those taken in the morning.
  5. Specific species of bacteria associated with individual metro lines. That is, sets of bug types are relatively abundant on a given line compared with all other lines, giving a kind of line-specific signature — though the distinction declines from morning to afternoon. The most physically isolated line, MOS (Ma On Shan), had a greater number of signature species. The MOS runs entirely above ground alongside the Shing Mun Channel, a polluted brackish river, and its ‘signature’ includes bacteria found in sewage.
  6. All of which brings us to bugs with antibiotic resistance genes (ARGs). Across the network 136 ARG families were detected including 24 that are clinically important. Strikingly, lines closer to Shenzhen (ER (East Rail) and MOS) tend to have higher ARG input during the day. Critically, the ER line a.m. signatures become p.m.-enriched in all MTR lines far from Shenzhen — that is, these ARG families spread over the network during the day.

Simplified map of the Hong Kong MTR indicating how antibiotic resistance genes spread during the day from the ER and MOS lines to the entire network. Tetracycline resistance genes: tetA, tetO, tetRRPP and tetMWOS; vancomycin resistance genes: vanC, vanX. From Kang et al. 2018.

These results clearly suggest that the ER line, the only cross-border line linked to mainland China, may be a source of clinically important ARGs, especially against tetracycline, a commonly used antibiotic in China’s swine feedlots. Antibiotics, including tetracycline, can be detected in the soil in the Pearl River Delta area where the cities of Hong Kong and Shenzhen are located.

It should be said that this is by no means the first survey of bugs on rails. Notable ones have looked at the New York and Boston metro systems and they too revealed the potential health risks of the bug communities found on trains and in the stations, including the presence of pathogens and antibiotic resistance. The Boston survey highlighted that different types of materials have surfaces that are preferred by different microbes with high variation in functional capacity and pathogenic potential.

One obvious suggestion from these studies is that world-wide we could do a lot to improve sanitation, e.g., by having hand sanitizer dispensers in all sensible places (at the exits of metro, railway and bike-sharing stations and airports and of course in hospitals). The Hong Kong data are seriously frightening and most people seem blissfully unaware that the invisible world they reveal carries the potential for the destruction of us all.

But, as ever, there’s two sides to the matter. We’ve evolved over millions of years to live with bugs and they with us. However you wash your hands you won’t get rid of every bug and anyway, as what’s-his-name almost says, “They’ll be back!” We all carry around micro-organisms that can be fatal if they get to the wrong place. But, if you’re reasonably fit, there’s a lot to be said for simply following sensible, basic hygiene rules with a philosophy of ‘live and let live.’

Have a nice day commuters, wherever you are!

References

Kang K., et al. (2018). The Environmental Exposures and Inner- and Intercity Traffic Flows of the Metro System May Contribute to the Skin Microbiome and Resistome. Cell Reports 24, 1190–1202.

Wu, N., Qiao, M., Zhang, B., Cheng, W.D., and Zhu, Y.G. (2010). Abundance and diversity of tetracycline resistance genes in soils adjacent to representative swine feedlots in China. Environ. Sci. Technol. 44, 6933–6939.

Li, Y.W., Wu, X.L., Mo, C.H., Tai, Y.P., Huang, X.P., and Xiang, L. (2011). Investigation of sulfonamide, tetracycline, and quinolone antibiotics in vegetable farmland soil in the Pearl River Delta area, southern China. J. Agric. Food Chem. 59, 7268–7276.

Leung, M.H., Wilkins, D., Li, E.K., Kong, F.K., and Lee, P.K. (2014). Indoor-air microbiome in an urban subway network: diversity and dynamics. Appl. Environ. Microbiol. 80, 6760–6770.

Robertson, C.E., Baumgartner, L.K., Harris, J.K., Peterson, K.L., Stevens, M.J., Frank, D.N., and Pace, N.R. (2013). Culture-independent analysis of aerosol microbiology in a metropolitan subway system. Appl. Environ. Microbiol. 79, 3485–3493.

Afshinnekoo, E., Meydan, C., Chowdhury, S., Jaroudi, D., Boyer, C., Bernstein, N., Maritz, J.M., Reeves, D., Gandara, J., Chhangawala, S., et al. (2015). Geospatial Resolution of Human and Bacterial Diversity with City-Scale Metagenomics. Cell Syst 1, 72–87.

Hsu, T., Joice, R., Vallarino, J., Abu-Ali, G., Hartmann, E.M., Shafquat, A., Du- Long, C., Baranowski, C., Gevers, D., Green, J.L., et al. (2016). Urban Transit System Microbial Communities Differ by Surface Type and Interaction with Humans and the Environment. mSystems 1, e00018–e00016.

Fantastic Stuff

 

It certainly is for Judy Perkins, a lady from Florida, who is the subject of a research paper published last week in the journal Nature Medicine by Nikolaos Zacharakis, Steven Rosenberg and their colleagues at the National Cancer Institute in Bethesda, Maryland. Having reached a point where she was enduring pain and facing death from metastatic breast cancer, the paper notes that she has undergone “complete durable regression … now ongoing for over 22 months.”  Wow! Hard to even begin to imagine how she must feel — or, for that matter, the team that engineered this outcome.

How was it done?

Well, it’s a very good example of what I do tend to go on about in these pages — namely that science is almost never about ‘ground-breaking breakthroughs’ or ‘Eureka’ moments. It creeps along in tiny steps, sideways, backwards and sometimes even forwards.

You may recall that in Self Help – Part 2, talking about ‘personalized medicine’, we described how in one version of cancer immunotherapy a sample of a patient’s white blood cells (T lymphocytes) is grown in the lab. This is a way of either getting more immune cells that can target the patient’s tumour or of being able to modify the cells by genetic engineering. One approach is to engineer cells to make receptors on their surface that target them to the tumour cell surface. Put these cells back into the patient and, with luck, you get better tumour cell killing.

An extra step (Gosh! Wonderful GOSH) enabled novel genes to be engineered into the white cells.

The Shape of Things to Come? took a further small step when DNA sequencing was used to identify mutations that gave rise to new proteins in tumour cells (called tumour-associated antigens or ‘neoantigens’ — molecular flags on the cell surface that can provoke an immune response – i.e., the host makes antibody proteins that react with (stick to) the antigens). Charlie Swanton and his colleagues from University College London and Cancer Research UK used this method for two samples of lung cancer, growing them in the lab to expand the population and testing how good these tumour-infiltrating cells were at recognizing the abnormal proteins (neo-antigens) on cancer cells.

Now Zacharakis & Friends followed this lead: they sequenced DNA from the tumour tissue to pinpoint the main mutations and screened the immune cells they’d grown in the lab to find which sub-populations were best at attacking the tumour cells. Expand those cells, infuse into the patient and keep your fingers crossed.

Adoptive cell transfer. This is the scheme from Self Help – Part 2 with the extra step (A) of sequencing the breast tumour. Four mutant proteins were found and tumour-infiltrating lymphocytes reactive against these mutant versions were identified, expanded in culture and infused into the patient.

 

What’s next?

The last step with the fingers was important because there’s almost always an element of luck in these things. For example, a patient may not make enough T lymphocytes to obtain an effective inoculum. But, regardless of the limitations, it’s what scientists call ‘proof-of-principle’. If it works once it’ll work again. It’s just a matter of slogging away at the fine details.

For Judy Perkins, of course, it’s about getting on with a life she’d prepared to leave — and perhaps, once in while, glancing in awe at a Nature Medicine paper that does not mention her by name but secures her own little niche in the history of cancer therapy.

References

McGranahan et al. (2016). Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 10.1126/science.aaf490 (2016).

Zacharakis, N. et al. (2018). Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nature Medicine 04 June 2018.