Transparently Obvious

 

Scientists have a well-earned reputation for doing odd things – by which I mean coming up with a ‘finding’ that leaves me, at least, wondering how, in the name of all things wonderful, they ever got money to do their study. To be fair, it’s the ‘social scientists’ – rather than the ‘real’ lot – that excel in this field. An example? Take your pick. They crop up pretty well weekly in the press. I liked the one on how something called ‘personal congruence’ affects marriage survival. The more congruence you and your partner have the better your chances: if, over time, your congruence goes down the tubes, your relationship will surely follow. But what on earth is congruence? Seemingly it’s a ‘state of agreeing.’ Lots of it equals harmony, loss of it = discord. So, it is what you remember from school geometry: it means more or less equal. Wow! Now I’ve grasped the upshot of this ‘study’: agreeably happy couples tend to make it: pairings based on whacking each other with frying pans tend to end in tears. Why didn’t they tell us earlier!!

Axolotl

   Axolotl

Fortunately, in my world, even the weirdies usually turn out to be quite sensible, once you know what’s going on. Many moons ago a girl-friend asked me if I’d like to see her collection of axolotls. Not having a clue what she was on about I gave it an excited ‘yes please’. Whilst it mayn’t have been what I was hoping for (I was very young back then), I immediately fell in love with these wonderful amphibians that I’d never heard of as she explained what I should have known: these ‘Mexican walking fish’ have very large embryos which makes them particularly useful for studying development. These sensational salamanders really are amazing, not least because they can regenerate entire limbs after they’ve been chopped off.

More recently there’s been another unlikely recruit to the scientific armoury: the zebrafish – a tropical freshwater fish from the Himalayas. This mighty minnow was the first vertebrate to be cloned which led to its being genetically modified to give a transparent variety. That’s all good fun but what on earth is the point of a see-through fish? Well, in Betrayed by Nature we pointed out that you can actually watch tumours growing in transparent zebrafish and we got so excited by that we even included a photo – kindly provided by Richard White of the Dana Farber Cancer Institute in Boston. The cancer was a melanoma which had grown into a black mass about 1 cm in diameter in the fish’s body after a small number of tumour cells had been injected a couple of weeks earlier.

And the driver is …

Nearly 15 years ago, just as the first complete sequence of human DNA was being unveiled, Mike Stratton and his colleagues at the Sanger Centre in Cambridge discovered a mutation that arises in about two-thirds of all malignant melanomas. It’s in a gene called BRAF. The protein made by the gene is an enzyme that’s part of a signalling pathway that pushes cells to divide. The mutation changes the shape of BRAF protein so it works 24/7 as an enzyme: the pathway is no longer controlled by a message from the world beyond the cell. It’s a ‘molecular switch’ that’s been flipped by mutation to act as a cancer ‘driver.’

Richard White and his colleagues showed that the same mutation drove melanoma development in zebrafish and that when it did so something remarkable happened. As the tumours got going they turned on a gene that is normally only required during the first 72 hours after fertilization. The gene’s called crestin – because it’s switched on in a tissue called the neural crest where crestin protein helps to form the bony support for the gills. After that it’s switched off and crestin protein never appears again. Except in the pigment-containing cells called melanocytes when they are turning into a tumour.

Seeing the problem

In a great example of how science can work, Charles Kaufman, Leonard Zon and colleagues in Boston and other centres took this finding and made another transgenic variant of the transparent zebrafish. They cut out the stretch of DNA that controls whether the crestin gene is ‘on’ or ‘off’ and hooked it up to a gene that makes a green fluorescent protein (GFP). Result: when the machinery of a cell turns crestin on, GFP is also made – and the cell glows green under the appropriate light. Hence you would expect to see a glowing neural crest early in development but thereafter a non-glowing fish. Unless it has a melanoma. And Zon & Co saw exactly that. Because green fluorescent protein glows so brightly, a single cell shows up and it turned out that whenever one green cell was detected it always went on to expand and grow into a large melanoma tumour.

1 cell to mel

Tracking a single cell turning into a tumour over 6, 9, 11.5 and 17 weeks. The green fluorescence marks an early developmental gene (crestin) being re-activated in a melanoma tumour (from Kaufman et al., 2016).

But why might it be useful to ‘see’ single cells?

Since the original finding by Stratton & Co more detailed studies have confirmed that mutated BRAF is indeed an important ‘driver’ in about two-thirds of malignant melanoma. But here’s the odd thing: lots of melanocytes (the cells that can turn into melanomas) have mutated BRAF – but they don’t become cancerous. Why not? And there’s something else: it’s well-known that ultraviolet radiation in sunlight causes many melanomas and they do indeed often arise on exposed skin – but they can also crop up in places where, as they say, the sun doesn’t shine. So clearly, important though mutated BRAF and sunlight are, there’s something else that’s critical for malignant melanoma.

The Kaufman experiment was remarkable, not least because it offers a way of getting at this key question of what happens in a cell to kick it off as a tumour, by comparison with a near neighbour that remains ‘normal.’

The tumour cells used in this model carry mutated BRAF and another gene, P53, was knocked out. This gives two major genetic drivers and it may be that further genetic changes aren’t needed. If that’s the case, then the decisive push must come either from epigenetic changes (that affect gene expression without change in DNA sequence) or from adaptations of the tumour microenvironment to provide an optimal niche for expansion. At the moment we don’t know very much about these critical areas of cancer biology. Being able to follow single cells may lead us to the answers.

Keep your eye on the transparent minnows!

Reference

Kaufman, C.K., Zon, L.I. et al. (2016). A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science 351, Issue 6272, pp. DOI: 10.1126/science.aad2197

 

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