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.

Advertisements

Guess Who’s Coming to Dinner?

 

Question: when is a gene not a gene? Answer: when it’s a pseudogene.

Genes are familiar enough these days when the acronym DNA has become part of everyday speech “It is in Toyota’s DNA that mistakes made once will not be repeated”, as the CEO of Toyota rather sinisterly remarked. You could say that’s pseudo-scientific rubbish but, despite that kind of liberty-taking, most will know that a gene is a stretch of our genetic material (DNA) that carries the code to make a closely related RNA molecule that, in turn, may be used as a template to make a protein ­– it’s the molecular unit of heredity. Well known too is that the Greeks gave us ‘pseudo’ – but what’s a ‘lying’ or ‘false’ gene – and who cares?

No prizes for guessing that we should all be interested because it’s emerging that pseudogenes can be important players in cancer.

Player’s biography

Pseudogenes are somewhat disreputable because they are relatives of normal genes that along the evolutionary highway have become dysfunctional by losing the capacity to be ‘expressed’ – that is, their code can no longer be transformed into RNA and protein. You could think of them as an example of the shambolic way in which species have evolved by random happenstance so that they work in their own particular niches. And if you want the outstanding example of unintelligent design, look no further than yourself, as we did in Holiday Reading (2), Poking the Blancmange.

Just for background, although it doesn’t affect the main story, there are three ways in which our genome can acquire a pseudogene:

1. A normal gene becomes functionally extinct: odd mutational events disable the stretches of DNA that control its expression. The gene is like a siding on a railway that isn’t used for years and years until eventually the points  seize up (it would be a ‘switch’ on US railroads) and the cell machinery can no longer get at it – but when this does happen we get by without that gene.

2. During evolution genes quite often get duplicated – giving multiple copies: if one of these loses its regulatory bits the duplicate gene is switched off – it’s become a ghost.

3. We owe about 8% of our genome to viruses – mainly those with RNA genomes (retroviruses) whose life-cycle turns their RNA into DNA that has then been stuck into our genome. And that’s a lot (about 100,000 bits of retrovirus DNA) especially bearing in mind that only about 1% of our genome encodes proteins.

So our precious genome is littered with corpses and fragments thereof. In the past there’s been a regrettable tendency to label this material as ‘junk’ but increasingly we’re now discovering that there may be genetic life after death, so to speak. It’s not surprising if you think about it. If random events can inactivate a gene then they might do the reverse, even if that may be a much rarer event. And indeed it’s now clear that pseudogenes can be brought back to life through the random mutational events that characterise the rough and tumble of cellular life.

So not all pseudogenes are extinct then?

Correct. Obviously we wouldn’t be wittering on about them had not some bright sparks just shown that pseudogenes – or at least one in particular – can be re-awakened to play a part in cancer. The luminaries are Florian Karreth, Pier Paolo Pandolfi and friends from all over the place (USA, UK, Italy, Singapore) who found that a pseudogene called BRAFP1 (a relative of the normal BRAF gene) can help to drive cancer development. Some earlier studies had shown that BRAFP1 was expressed (i.e. RNA was made from DNA) in various human tumours but Karreth & Co extended this, detecting significant levels of the pseudogene RNA in lymphomas and thyroid tumours and also in cells from melanoma, prostate cancer and lung cancer, whilst it’s not switched on in the corresponding normal cells.

To show that this pseudogene can drive cancers they genetically engineered its over-expression in mice, whereupon the animals developed an aggressive malignancy akin to human lymphoma (specifically diffuse large B cell lymphoma). Short-circuiting an enormous amount of work, it emerged that the pseudogene up-regulated a signaling pathway involving its normal counterpart, BRAF, that drives proliferation.

106 pic

How a pseudogene (BRAFP1) might drive cancer. Top: The scheme illustrates the ‘central dogma’ of molecular biology: DNA makes RNA makes protein. In normal cells a family of micro RNAs (different coloured wiggles) regulate the level of BRAF RNA and hence of BRAF protein (above white line).  Bottom: When the pseudogene BRAFP1 is switched on its RNA competes for the negative regulators: the result is more BRAF RNA making more BRAF protein – making cancer (Karreth et al., 2015).

Interfering RNA

The pseudogene’s RNA manages to interfere with normal control by targeting another type of RNA – micro RNAs, so called because they’re very short (about 20 bases (units) long – so they’re encoded by tiny stretches of the over 3,000 million units that make up the genome). Small they may be but there are hundreds of them and it’s become clear over the last few years that they play critical roles in regulating how much protein is made from specific RNAs. Their method is simple: they recognize (i.e. bind to) stretches of RNA that encode proteins, thereby blocking translation into protein.

Karreth & Co showed that there are about 40 different micro RNAs that can stick to the RNAs encoding BRAF or BRAFP1. Normally when there’s no (or very little) BRAFP1 around they have only BRAF to act on – and their role is to control the proliferation signal it transmits – i.e. to keep that signal to what’s required for normal cell growth control. BUT, when the pseudogene RNA is made in significant amounts the attentions of the 40 micro RNAs are divided. Result: more BRAF RNA, more BRAF protein, higher cell proliferation.

It’s a bit like you’re just sitting down to a family dinner for four when there’s a knock on the door and in walks long lost Uncle Bert, complete with wife and two kids in tow. Of course you invite them to dine too – but now a meal for four has to stretch to eight. There is something for everybody – just not as much. Similarly for the regulators of BRAF: when BRAFP1 is present there’s half as much of the RNA regulators for each – and the result, bearing mind that they are negative regulators, is that the activity of BRAF goes up and the cells proliferate more avidly. The pseudogene is driving cancer.

First but not last

For decades pseudogenes were thought of as ‘junk’ DNA along with most of the rest of the genome that didn’t encode proteins – though I might say that was a concept I never promoted. Beware labeling anything in our genome as junk for it may rise, Kraken like, to remind us of our ignorance. And, now that one pseudogene has come in from the cold and been shown to drive some cancers, you can be confident that others will follow.

References

Karreth, F.A. et al. (2015). The BRAF Pseudogene Functions as a Competitive Endogenous RNA and Induces Lymphoma In Vivo. Cell 161, 319–332.