Be amazed

 

Back in May 2018 we reported the first output from the Pan-Cancer Atlas, a massive undertaking that evolved from The Cancer Genome Atlas, itself a huge project aiming to set up a genetic data-base for three cancer types: lung, ovarian, and glioblastoma.

The next instalment from the Pan-Cancer Analysis of Whole Genomes (PCAWG) has just appeared featuring the analysis of a staggering 2,658 whole-cancer genomes and their matching, normal tissues across 38 tumour types and it has reminded us, yet again, of nature’s capacity to surprise. The first finding was that, on average, cancer genomes contained four or five driver mutations when coding and non-coding genomic elements were combined. That’s roughly consistent with the accepted estimate over the last few decades. What was unexpected, however, was that in around 5% of cases no drivers were identified, suggesting that there are more of these mutations to be discovered. Also somewhat surprising is that chromothripsis, the single catastrophic event producing simultaneously many variants in DNA, is frequently an early event in tumour evolution.

The analyses also revealed several mechanisms by which the ends of chromosomes in cancer cells are protected from telomere attrition and that variants transmitted in the germline can affect subsequently acquired patterns of somatic mutation.

A glimpse of the data

The panorama of driver mutations includes the summary below of tumour-suppressor genes with biallelic inactivation (i.e., mutation of one allele (copy) followed by gene deletion of the remaining allele) in 10 or more patients. Familiar tumour suppressors are prominent on the left hand side, as expected. These include TP53 (the guardian of the genome) and the tumour suppressors CDKN2A and CDKN2B (cyclin-dependent kinase inhibitors 2A and 2B) that regulate the cell cycle.

Tumour-suppressor genes for which both copies of the gene (alleles) are inactivated in 10 or more patients. GR = genomic rearrangement, i.e. chromosome breakage. From The ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium.

Aneuploidy in the genome of a tumour without known drivers. Each row is an individual tumour: the boxes show chromosome loss (blue) or gain (red). The cancer is a rare kidney tumour (chromophobe renal cell carcinoma). From The ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium.

Two tumour types had a surprisingly high fraction of patients without identified driver mutations: 44% for a rare type of kidney cancer (chromophobe renal cell carcinoma) and 22% in a rare pancreatic neuroendocrine cancer. It turned out (as shown in the above figure) that there was a striking loss or gain of chromosomes — called aneuploidy — in the cells of these cancers. This suggests that wholesale loss of tumour suppressor genes or gain of oncogenic function was providing the ‘drivers’ for these cancers.

The genomic cancer message

We should first acknowledge the mind-boggling effort and organization involved in collecting thousands of paired samples, sequencing them and analyzing the output. However, the value of these massive projects is beginning to emerge — and the news is mixed.

One critical trend is that genomic analysis is re-defining the way cancers are classified. Traditionally they have been grouped on the basis of the tissue of origin (breast, bowel, etc.) but this will gradually be replaced by genetic grouping, reflecting the fact that seemingly unrelated cancers can be driven by common pathways.

Perhaps the most encouraging thing to come out of the genetic changes driving these tumours is that for about half of them potential treatments are already available. That’s quite a surprise but it doesn’t mean that hitting those targets will actually work as anti-cancer strategies. Nevertheless, it’s a cheering point that the output of this phenomenal project may, as one of the papers noted, serve as a launching pad for real benefit in the not too distant future.

On the other hand, the intention of precision medicine is to match patients to therapies on the basis of genomics and, notwithstanding the above point, the consortium notes that “A major barrier to evidence-based implementation is the daunting heterogeneity of cancer chronicled in these papers, from tumour type to tumour type, from patient to patient, from clone to clone and from cell to cell. Building meaningful clinical predictors from genomic data can be achieved, but will require knowledge banks comprising tens of thousands of patients with comprehensive clinical characterization. As these sample sizes will be too large for any single funding agency, pharmaceutical company or health system, international collaboration and data sharing will be required.”

See for yourself

The PCAWG landing page (http://docs.icgc.org/pcawg/) provides links to several data resources for interactive online browsing, analysis and download of PCAWG data.

Reference

The ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium. Pan-cancer analysis of whole genomes.

 

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.

Mushrooming Secret Army

 

We have in these pages talked quite a bit about our ‘secret army’ — the bugs that share our body to the extent that bacteria outnumber us on a cell-to-cell basis by at least three to one. As we noted in Secret Army: More Manoeuvres Revealed, bacteria are just one part of what is collectively called the microbiota’ but with over 2000 different species and a total gene pool hundreds of times bigger than our own 20,000 or so, they are by far the biggest. And it’s gradually become clear that they are not with us just because our bodies are warm, damp and comfortable but they help us get the most out of our food and they’re important in the working of our immune system.

Bacteria and cancer

Most critically, in the present context, we now know that shifts in proportions of species in the microbiome can influence cancer development and perhaps even the spread of tumour cells around the body.

Small fry

Important though they are, bacteria aren’t the only members of the microbiome — which includes fungi, viruses and various single-celled parasites (protozoa). Today’s story is about fungi, a group of microorganisms familiar to gardeners world-wide, that includes yeasts and molds, as well as the more familiar mushrooms. There’s estimated to be several million species of fungi, although only about 120,000 have been described. Some we can eat, some can kill us and, of course, there’s magic mushrooms.

With all this diversity you might wonder whether any fungi have elbowed their way into us to share the delights of the human body alongside bacterial microbes. Of course they have: most people will have heard of candidiasis — a fungal infection caused by Candida yeasts that belong to the genus Candida. Candida normally finds its niche in places like the mouth (giving the condition called thrush), gut, vagina and on the skin and usually doesn’t give us any trouble. But, truth to tell, we’ve known very little about fungi in us until recently when the power of DNA sequencing has started to be applied to the topic. This has confirmed that we do carry lots of fungi around with us, albeit that they are only a tiny fraction of the microbial community (somewhat less than 0.1%).

New actor in the cancer cast

This fungal force of microbes is known as the mycobiome (as distinct from the microbiome) and, in contrast to bacteria, there is no evidence that it has a role in cancer. Until, that is, the recent publication from New York University School of Medicine by Berk Aykut, George Miller and friends showing that fungi travel from the gut to the pancreas where a particular species can actually give cancer a helping hand. The cancer in question is pancreatic ductal adenocarcinoma (PDA) that has a particularly dismal prognosis.How a fungus can drive cancer. The scheme represents a tumour in the pancreas changing the make up of the adjacent fungal community and how a protein in the blood called mannose binding lectin (MBL) can attach to the outer surface of a fungal cell. When this happens MBL changes shape so it can then stick to another protein (C3) which in turn activates a relay of proteins called the complement cascade. One upshot of this can be to promote tumour growth. From Dambuza and Brown 2019.

How did they do it?

Aykut et al. first used DNA sequencing to look for fungus-specific sequences in the pancreas of humans with PDA and in mouse models of PDA, They’d previously shown that the bacterial load goes up by about 1000-fold in tumours compared with healthy tissue and, lo and behold, they found a similar increase in fungi. Next they tagged strains of fungus with a fluorescent label and showed that the cells could migrate from the gut to the pancreas of mice in under 30 minutes.

They then tracked down a protein called mannose binding lectin (MBL) expression of which is associated with poor survival in human PDA patients. MBL is a ‘serum protein’, meaning that it floats around in blood. This led to the discovery that MBL can bind to the surface of fungal cells and when it does so changes shape to permit activation of a relay of signal proteins called the complement system. This ‘complement cascade’ is part of our immune system, enhancing the capacity of antibodies and phagocytic cells to clear microbes from the circulation.

Jules Bordet was the chap who first showed that something in normal blood plasma could help to kill off bacteria back at the end of the 19th century and, as such, deserves to be better remembered as a famous Belgian.

The complement system is pretty amazing because, whilst it can trigger an immune response against invading pathogens, it can also switch on inflammatory pathways that help cells grow and move around — in other words, give a helping hand to tumours.

Fungible?

I met this word for the first time a few days ago, courtesy of the journalist and author Ann Treneman. You’d think that no piece on fungi would be complete without it but it turns out to have nothing to do with mushrooms: it just means interchangeable or switchable. But hang on! We can squeeze it in by asking a very relevant question: are pancreatic fungi fungible in terms of their capacity to promote cancer? Aykut et al. did just that and the answer was ‘no they’re not.’ One species seems to be particularly abundant in PDA: the genus Malassezia. This was true for both mouse and human tumours and perhaps that shouldn’t surprise us as Malassezia is the most abundant fungal species in mammalian skin, accounting for more than 80% of our skin mycobiome. So it’s Malassezia not other species (e.g., Candida) that has the power to drive cancer.

Spores of the yeast Malassezia

Fungal footnote

In a final exciting experiment Aykut et al. showed that antifungal drugs halted PDA progression in mice and improved the ability of chemotherapy to shrink the tumour. This obviously raises the notion that if we can find ways of shifting the balance of fungal communities or interfering with the link to the complement cascade we might have a completely new line on desperately needed therapies for this disease.

References

Aykut, B. et al., (2019). The fungal mycobiome promotes pancreatic oncogenesis via activation of MBL. Nature 574, 264–267.

Dambuza, I.M. and Brown, G.D. (2019). Fungi accelerate pancreatic cancer. Nature 574, 184-185.

Secret Army: More Manoeuvres Revealed

 

I don’t know about you but I find it difficult to grasp the idea that there are more bugs in my body than there are ‘me’ cells. That is, microorganisms (mostly bacteria) outnumber the aggregate of liver, skin and what-have-you cells. They’re attracted, of course, to the warm, damp surfaces of the cavities in our bodies that are covered by a sticky, mucous membrane, e.g., the mouth, nose and especially the intestines (the gastrointestinal tract).

The story so far

Over the last few years it’s become clear that these co-residents — collectively called the microbiota — are not just free-loaders. They’re critical to our well-being in helping to fight infection by other microrganisms (as we noted in Our Inner Self), they influence our immune system and in the gut they extract the last scraps of nutrients from our diet. So maybe it makes them easier to live with if we keep in mind that we need them every bit as much as they depend on us.

We now know that there are about 2000 different species of bacteria in the human gut (yes, that really is 2,000 different types of bug) and, with all that diversity, it’s not surprising that the total number of genes they carry far exceeds our own complement (by several million to about 20,000). In it’s a small world we noted that obesity causes a switch in the proportions of two major sub-families of bacteria, resulting in a decrease in the number of bug genes. The flip side is that a more diverse bug population (microbiome) is associated with a healthy status. What’s more, shifts of this sort in the microbiota balance can influence cancer development. Even more remarkably, we saw in Hitchhiker Or Driver? That the microbiome may also play a role in the spread of tumours to secondary sites (metastasis).

Time for a deep breath

If all this is going on in the intestines you might well ask “What about the lungs?” — because, and if you didn’t know you might guess, their job of extracting oxygen from the air we inhale means that they are covered with the largest surface area of mucosal tissue in the body. They are literally an open invitation to passing microorganisms — as we all know from the ease with which we pick up infections.

In view of what we know about gut bugs a rather obvious question is “Could the bug community play a role in lung cancer?” It’s a particularly pressing question because not only is lung cancer the major global cause of cancer death but 70% lung cancer patients have bacterial infections and these markedly influence tumour development and patient survival. Tyler Jacks, Chengcheng Jin and colleagues at the Massachusetts Institute of Technology approached this using a mouse model for lung cancer (in which two mutated genes, Kras and P53 drive tumour formation).

In short they found that germ-free mice (or mice treated with antibiotics) were significantly protected from lung cancer in this model system.

How bacteria can drive lung cancer in mice. Left: scheme of a lung with low levels of bacteria and normal levels of immune system cells. Right: increased levels of bacteria accelerate tumour growth by stimulating the release of chemicals from blood cells that in turn activate cells of the immune system to release other effector molecules that promote tumour growth. The mice were genetically altered to promote lung tumour growth (by mutation of the Kras and P53 genes). In more detail the steps are that the bacteria cause macrophages to release interleukins (IL-1 & IL-23) that stick to a sub-set of T cells (γδ T cells): these in turn release factors that drive tumour cell proliferation, including IL-22. From Jin et al. 2019.

As lung tumours grow in this mouse model the total bacterial load increases. This abnormal regulation of the local bug community stimulates white blood cells (T cells present in the lung) to make and release small proteins (cytokines, in particular interleukin 17) that signal to neutrophils and tumour cells to promote growth.

This new finding reveals that cross-talk between the local microbiota and the immune system can drive lung tumour development. The extent of lung tumour growth correlated with the levels of bacteria in the airway but not with those in the intestinal tract — so this is an effect specific to the lung bugs.

Indeed, rather than the players prominent in the intestines (Bs & Fs) that we met in Hitchhiker Or Driver?, the most common members of the lung microbiome are Staphylococcus, Streptococcus and Lactobacillus.

In a final twist Jin & Co. took bacteria from late-stage tumours and inoculated them into the lungs of mice with early tumours that then grew faster.

These experiments have revealed a hitherto unknown role for bacteria in cancer and, of course, the molecular signals identified join the ever-expanding list of potential targets for drug intervention.

References

Jin, C. et al. (2019). Commensal Microbiota Promote Lung Cancer Development via γδ T Cells. Cell 176, 998-1013.e16.

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.

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.

Turning Ourselves On

 

It may seem a bit tasteless but we have to admit that cancer’s a very ‘trendy’ field. That is, there’s always a current fad — something for which either the media or cancer scientists themselves have the hots. Inevitable I suppose, given the importance of cancer to pretty well everyone and the fact that something’s always happening.

If you had to pick the front-running trends of late I guess most of us might go for ‘personalized medicine’ and ‘immunotherapy.’ The first means tailoring treatment to the individual patient, the second is boosting the innate power of the immune system to fight cancer.

Few things are trendier than this blog so it goes without saying that we’ve done endless pieces on these topics (e.g. Fantastic Stuff, Outsourcing the Immune Response, Self-Help – Part 2, bla, bla, bla).

How considerate then of Krijn Dijkstra, Hans Clevers, Emile Voest and colleagues from the Netherlands Cancer Institute to have neatly combined the two in their recent paper.

Simple really

What they did was did was easy — in principle. They grew fresh tumour tissue from patients in dishes in the laboratory. Although it doesn’t work every time, most of the main types of cancer have been grown in this way to give 3D cultures called tumour organoids — tumours-in-a-dish. That’s the ‘personalized’ bit.

Then they took blood from the patient and grew the lymphocytes therein in a dish to expand the T cells that were specific for the patient’s tumour. That’s the ‘‘immuno’ bit.

Growing tumour tissue (from non-small-cell lung cancer (NSCLC) and colorectal cancers [CRC] in culture as tumour organoids. This permits the expansion of T cells from peripheral blood to give an enlarged population of cells that will kill those tumours. From Dijkstra et al. 2018.

And the results?

They were able to show that enriched populations of tumour-reactive T cells could kill tumour organoids and, importantly, that organoids formed from healthy tissue were not attacked by these T cells.

Stained organoids (left) and original tissue (right) from two colorectal cancers (CRC-2 & CRC-5) showing how the organoids grow to have an architecture similar to the original tumour. From Dijkstra et al. 2018.

Their method worked for both bowel tumours and non-small-cell lung cancer but there’s no reason to suppose it can’t be extended to other types of cancer.

Some of their videos showing tumour organoids being chomped up by enriched killer T cells are quite dramatic. Cells labelled green that can be seen in this video are dying.

So there you have it: DIY tumour therapy!

Reference

Dijkstra, K.K. et al. (2018). Generation of Tumor-Reactive T Cells
by Co-culture of Peripheral Blood Lymphocytes and Tumor Organoids. Cell 174, 1–13.