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.

The Power of Flower

 

We know we don’t ‘understand cancer’ — for if we did we would at least be well on the way to preventing the ten million annual deaths from these diseases and perhaps even stymieing their appearance in the first place. But at least, after many years of toil by thousands of curious souls, we might feel brave enough to describe the key steps by which it comes about.

Here goes!

Our genetic material, DNA, carries a code of four different units (bases) that enables cells to make twenty-thousand or so different types of proteins. Collectively these make cells — and hence us — ‘work’. An indicator of protein power is that we grow from single, fertilized cells to adults with 50 trillion cells. That phenomenal expansion involves, of course, cells growing and dividing to make more of themselves — and, along the way, a bit of cell death too. The fact that there are nearly eight billion people on planet earth testifies to the staggering precision with which these proteins act.

Nobody’s perfect

As sports fans will know, the most successful captain in the history of Australian rugby, John Eales, was nicknamed ‘Nobody’ because ‘Nobody’s perfect’. Well, you might care to debate the infallibility of your sporting heroes but when it comes to their molecular machinery, wondrous though it is, perfect it is not.

Evidence: from the teeming eight billion there emerges every year 18 million new cancer cases (that’s about one in every 444). And cancers are, of course, abnormal cell growth: cells growing faster than they should or growing at the wrong time or in the wrong place — any of which means that some of the masterful proteins have suffered a bit of a malfunction, as the computer geeks might say.

How can that happen?

Abnormal protein activity arises from changes in DNA (mutations) that corrupt the normal code to produce proteins of greater or lesser activity or even completely novel proteins.

These mutations may be great or small: changes in just one base or seismic fragmentation of entire chromosomes. But the key upshot is that the cell grows abnormally because regulatory proteins within the cell have altered activity. Mutations can also affect how the cell ‘talks’ to the outside world, that is, the chemical signals it releases to, for example, block immune system killing of cancer cells.

Clear so far?

Mutations can change how cells proliferate, setting them free of normal controls and launching their career as tumour cells and, in addition, they can influence the cell’s environment in favour of unrestricted growth.

The latter tells us that cancer cells cooperate with other types of cell to advance their cause but now comes a remarkable discovery of a hitherto unsuspected type of cellular collaboration. It’s from Esha Madan, Eduardo Moreno and colleagues from Lisbon, Arkansas, St. Louis, Indianapolis, Omaha, Dartmouth College, Zurich and Sapporo who followed up a long-known effect in fruit flies (Drosophila) whereby the cells can self-select for fitness to survive.

Notwithstanding the fact that flies do it, the idea of a kind of ‘cell fitness test’ is novel as far as human cells go — but it shouldn’t really surprise us, not least because our immune system (the adaptive immune system) features much cooperation between different types of cell to bring about the detection and removal of foreign or damaged cells.

Blooming science

So it’s been known for over forty years that Drosophila carries out cell selection based on a ‘fitness fingerprint’ that enables it to prevent developmental errors and to replace old tissues with new. However, it took until 2009 before the critical protein was discovered and, because mutant forms of this protein gave rise to abnormally shaped nerve cells that looked like bunches of flowers, Chi-Kuang Yao and colleagues called the gene flower‘.

Cells can make different versions of flower proteins (by alternative splicing of the gene) the critical ones being termed ‘winner’ and ‘loser’ because when cells carrying winner come into contact with cells bearing loser the latter die and the winners, well, they win by dividing and filling up the space created by the death of losers.

The effect is so dramatic that Madan and colleagues were able to make some stunning movies of the switch in cell populations that occured when they grew human breast cancer cells engineered to express different version of flower tagged with red or green fluorescent labels.

Shift in cell populations caused by two types of flower proteins. 

Above are images at time zero and 24 h later of co-cultures of cells expressing  green and red proteins (losers and winners). From Madan et al. 2019.

Click here to see the movie on the Nature website.

Winner takes almost all

The video shows high-resolution live cell imaging over a 24 hour period compressed into a few seconds. Cells expressing the green protein (hFwe1 (GFP)) were co-cultured with red cells (hFwe2 (RFP)). Greens are losers, reds winners. As the movie progresses you can see the cell population shifting from mainly green to almost entirely red, as the first and last frames (above) show.

How does flower power work?

Flower proteins form channels across the outer membrane of the cell that permit calcium flow, and it was abnormal calcium signalling that caused flowers to bloom in Drosophila nerves. It would be reasonable to assume that changes in calcium levels are behind the effects of flower on cancer cells. Reasonable but wrong, for Madan & Co were able to rule out this explanation. At the moment we’re left with the rather vague idea that flower proteins mediate competitive interactions between cells and these determine whether cells thrive and proliferate or wither and die.

Does this really happen in human cancers?

Madan and colleagues looked at malignant and benign human tumours and found that there was more ‘winner’ flower protein in the former than the latter and that ‘loser’ levels were higher in normal cells next to a tumour than further away. Both consistent with the notion that tumour cells express winner and this induces loser in nearby normal cells leading to their death. What’s more they reproduced this effect in mice by transplanting human breast cancer cells expressing winner.

So there we are! After all this time a variant on how cancer cells can manipulate their surroundings to promote the development of tumours. Remarkable though this finding is, in a way that is familiar it’s just the beginning of this story. We don’t know how flower proteins work in giving cancers a helping hand and we don’t know how effective they are. Until we answer those questions it would be premature to try to come up with therapies to block their effect.

But it is a moment to sit back and reflect on the astonishing complexity of living organisms and their continuing capacity to surprise.

Reference

Madan, E. et al. (2019). Flower isoforms promote competitive growth in cancer. Nature 572, 260-264.

Yao, C-K., et al., (2009). A synaptic vesicle-associated Ca2+ channel promotes endocytosis and couples exocytosis to endocytosis. Cell 138, 947–960.

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.

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.

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.

I Know What I Like

 

I guess most of us at some time or other will have stood gazing at a painting for a while before muttering ‘Wow, that’s awesome’ or words to that effect if we’re not into the modern argot. Some combination of subject, style and colour has turned our crank and left us thinking we wouldn’t mind having that on our kitchen wall.

Given the thousands of years of man’s daubing and the zillions of forms that have appeared from pre-historic cave paintings through Eastern painting, the Italian Renaissance, Impressionism, Dadaism and the rest to Pop Art, it’s amazing that everyone isn’t a fanatic for one sort or another. The sane might say the field’s given itself a bad name by passing off tins of baked beans, stuff thrown at a canvas and unmade beds as ‘art’ but, even so, it seems odd that it remains a minority obsession.

Can science help?

Science is wonderful, as we all know, but the notion that it might arouse the collective artistic lust seems fanciful. Nevertheless, unnoticed by practically everyone, our vast smorgasbord of smears has been surreptitiously joined over the last 30 years by a new form: an ever-expanding avalanche of pics created by biologists trying to pin down how animals work at the molecular level. The crucial technical development has been the application of fluorescence in the life sciences: flags that glow when you shine light on them and can be stuck on to molecules to track what goes on in cells and tissues. The pioneer of this field was Roger Tsien who died, aged 64, in 2016.

Because this has totally transformed cell biology we’ve run into lots of brilliant examples in these pages — recently in Shifting the Genetic Furniture, in Caveat Emptor and John Sulston: Biologist, Geneticist and Guardian of our Heritage and in the use of red and green tags for picking out individual types of proteins that mark mini-cells within cells in Lorenzo’s Oil for Nervous Breakdowns.

To mark the New Year this piece looks at science from a different angle by focussing not on the scientific story but on the beauty that has become a by-product of this pursuit of knowledge.

Step this way: entrance free

So let’s take a stroll through our science gallery and gaze at just a few, randomly selected works of art.

  1. Cells grown in culture:

This was one of the first experiments in my laboratory using fluorescently labelled antibodies, carried out by a student, Emily Hayes, so long ago that she now has a Ph.D., a husband and two children. The cells are endothelial cells (that line blood vessels). Blue: nuclei; green: F-actin; red: Von Willebrand factor, a protein marker for endothelium.

 

  1. Two very recent images taken by my colleague Roderik Kortlever of a senescent mouse fibroblast and of mouse breast tissue:

 

 

 

 

 

 

3. Waves of calcium in firing neurons:

One of my fondest memories is helping to do the first experiment that measured the level of calcium within a cell, carried out with my colleague the late Roger Tsien and two other friends. I only grew the cells: Roger had designed and made the molecule, quin2. We didn’t know it at the time but Roger’s wonder molecule was the first of many intracellular ‘reporters.’ Roger shared the 2008 Nobel Prize in Chemistry for his discovery and development of the green fluorescent protein with organic chemist Osamu Shimomura and neurobiologist Martin Chalfie.

This wonderful video of a descendant of quin2 in nerve cells was made in Dr. Sakaguchi’s lab at Iowa State University.

 

4. Calcium wave flooding a fertilized egg: Taro Kaneuchi and colleagues at the Tokyo Metropolitan University:

Click for a time-lapse movie of an egg cell that has been artificially stimulated to show the kind of calcium change that happens at fertilization. In this time-lapse movie the calcium level reaches a maximum signal intensity after about 30 min before gradually decreasing to the basal level.

 

5. The restless cell (1):

This movie shows how protein filaments in cells can continuously break down and reform – called treadmilling. Visualised in HeLa cells using a green fluorescent protein that sticks to microtubules (tubular polymers made up of the protein tubulin) by HAMAMATSU PHOTONICS.

 

6. The restless cell (2):

This movie shows how mitochondria (organelles within the cell) are continuously changing shape and moving within the cell’s interior (cytosol). Red marks the mitochondria; green DNA within the nucleus. HAMAMATSU PHOTONICS.

 

7. Cell division:

Pig kidney cells undergoing mitosis. Red marks DNA (nucleus); green is tubulin: HAMAMATSU PHOTONICS.

 

8. DNA portrait of Sir John Sulston by Marc Quinn commissioned by the National Portrait Gallery:This image looks a bit drab in the present context but in some ways it’s the most dramatic of all. John Sulston shared the 2002 Nobel Prize in Physiology or Medicine with Sydney Brenner and Robert Horvitz for working out the cell lineage of the roundworm Caenorhabditis elegans (i.e. how it develops from a single, fertilized egg to an adult). He went on to sequence the entire DNA of C. elegans. Published in 1998, it was the first complete genome sequence of an animal — an important proof-of-principle for the Human Genome Project that followed and for which Sulston directed the British contribution at the Sanger Centre in Cambridgeshire, England. The project was completed in 2003.

The portrait shows colonies of bacteria in a jelly that, together, carry all Sulston’s DNA. This represents DNA cloning in which DNA fragments, taken up by bacteria after insertion into a circular piece of DNA (a plasmid), are multiplied to give many identical copies for sequencing.

 

9. “Brainbow” mice by Tamily Weissman at Harvard University:

The science behind this astonishing image builds on the work of Roger Tsien. Mice are genetically engineered to carry three different fluorescent proteins corresponding to the primary colours red, yellow and blue. Within each cell recombination occurs randomly, giving rise to different colours. The principle of mixing primary colours is the same as used in colour televisions.  In this view individual neurons in the brain (specifically a layer of the hippocampus) project their dendrites into the outer layer. Other magnificent pictures can be seen in the Cell Picture Show.

It’s certainly science – but is it art?

A few years ago the Fitzwilliam Museum in Cambridge staged Vermeer’s Women, an exhibition of key works by Johannes Vermeer and over thirty other masterpieces from the Dutch ‘Golden Age’. I tried the experiment of standing in the middle of each room and picking out the one painting that, from a distance, most caught my amateur eye. Funny thing was: not one turned out to be by the eponymous star of the show! Wondrous though Vermeer’s paintings were, the ones that really took my fancy were by Pieter de Hooch, Samuel van Hoogstraten and Nicolaes Maes, guys I’d never heard of.

Which made the point that you don’t need to be a big cheese to make a splash and that in the new Dutch Republic of the 17th century, the most prosperous nation in Europe, there was enough money to keep a small army of splodgers in palettes and paint. Skillful and incredibly patient though these chaps were, they simply used the tools available to paint what they saw in the world before them — as for the most part have artists down the ages.

But hang on! Isn’t that what we’ve just been on about? Scientists applying enormous skill and patience in using the tools they’ve developed to visualize life — to image what Nature lays before them. So the only difference between the considerable army of biological scientists around the world making a new art form and the Old Masters is that the newcomers are unveiling life — as opposed to the immortalizing a rather dopy-looking aristocrat learning to play the virginal or some-such.

Controversial?

Not really. Let’s leave the last word to Roger Tsien. In our final picture there are eight bacterial colonies each expressing a different colour of fluorescent protein arranged to grow as a San Diego beach scene in a Petri dish. It became the logo of Roger’s laboratory.

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.

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.