Back to Cell Biology

Because you’ve just gotta love cells. And because this post is about a publication in The Journal of Cell Biology, published by the Rockefeller University Press. In the summer of 2014 I spent almost three months doing an undergraduate research programme at Cold Spring Harbor Laboratory in the lab of Lloyd Trotman and under the everyday supervision of Dawid G. Nowak. I mainly helped Dawid establish the CRISPR/Cas9 method in the lab to study several types of cancers, including lung and prostate cancer. The first story, in which we used CRISPR to knockout a potent oncogene called Myc, was published almost two years ago (Nowak et al, 2015). Now Dawid is the co-first author on a new paper studying a tumour suppressor protein called PTEN (Chen, Nowak, … Wang, … et al, 2017).

Here is an eLife-style digest of the manuscript. Tumours usually evolve when cells gain the function of so-called oncogenes and lose the function of one or more so-called tumour suppressor genes. One of the most frequently deleted or down-regulated tumour suppressors is a protein called PTEN. Some cancer types, including some types of lung and prostate cancer, do not always delete the two gene copies coding for the PTEN protein, but the levels of PTEN protein in those cancer cells is still kept low. Therefore we wanted to find out which pathways in cancer cells lower the PTEN protein levels. Knowing about this regulation could lead to the development of new therapies that aim at stabilising PTEN protein.

First, we used both mouse and human cancer cell lines to investigate the movement of PTEN between the cytoplasm and the nucleus. We hypothesised that PTEN might be protected from being degraded in the nucleus, since the enzymes that break proteins down are generally found in the cytoplasm. Biochemical experiments showed that PTEN was moved into the nucleus by a protein called importin-11. Next, and this is the experiment I performed, we deleted importin-11 using CRISPR/Cas9 and saw that PTEN abundance decreased, while active/phosphorylated Akt, an oncogene, increased:

screen-shot-2017-02-12-at-16-20-15

Western blot showing CRISPR/Cas9 deletion of importin-11 in human prostate cancer cell lines – copied directly from Fig. 2 of Chen, Nowak et al, 2017

Further experiments conducted in the cell lines supported the following model, in which PTEN is shuttled into the nucleus by importin-11 where it is protected from degradation by the ubiquitin ligase system:

screen-shot-2017-02-12-at-16-20-37

Model of PTEN shuttling: when importin-11 is present PTEN can “hide” in the nucleus (left), but when importin-11 is deleted/not functioning, PTEN accumulates in the cytoplasm where it can be targeted for degradation – copied directly from Fig. 4 of Chen, Nowak et al, 2017

Next we wanted to know whether this mechanism of keeping levels of PTEN low is also important for preventing tumours. When importin-11 was experimentally down-regulated in mice (the gene for importin-11 was not completely deleted but its mutation is said to be “hypomorphic”), the mice developed and eventually died from lung cancers, unlike the healthy control mice:

screen-shot-2017-02-12-at-16-20-50

Lesion-free survival curve of importin-11 mutant (red) versus control (black) mice – copied directly from Fig. 5 of Chen, Nowak et al, 2017

Similar results were also obtained for prostate tumours in mice. Lastly, we analysed publicly available data of human prostate cancer patients. Low levels of importin-11 (either by genetic deletion or low gene expression) correlated with higher rates of tumour recurrence, suggesting that importin-11 also acts as a tumour suppressor in some types of human cancer. Future experiments may involve conducting more sophisticated mouse experiments in which importin-11 is deleted in specific organs, together with the activation of known oncogenes. This work may also lead to studies that try to find ways of stabilising PTEN protein.


So that’s it. Publication number three! But I want to end on a slightly more philosophical/political note. Dawid, one of the two first authors, taught me a lot during that summer programme, has been supportive ever since, and I enjoy keeping in touch with him. At the moment he is looking for an independent research position – he is enthusiastic about science and very driven. He’s had interviews all over the place, both in Europe and North America. However, Dawid is Polish and is now having to re-think his options since neither the UK nor the USA seem particularly appealing places for him anymore. We live in a crazy world but I hope this won’t stop him from getting the lab he deserves, in the most tolerant place possible.


References:

Chen M, Nowak DG, Narula N, Robinson B, Watrud K, Ambrico A, Herzka TM, Zeeman ME, Minderer M, Zheng W, Ebbesen SH, Plafker KS, Stahlhut C, Wang VMY, Wills L, Nasar A, Castillo-Martin M, Cordon-Cardo C, Wilkinson JE, Powers S et al. (2017) The nuclear transport receptor Importin-11 is a tumor suppressor that maintains PTEN protein. The Journal of Cell Biology DOI: 10.1083/jcb.201604025

Nowak DG, Cho H, Herzka T, Watrud K, DeMarco DV, Wang VM, Senturk S, Fellmann C, Ding D, Beinortas T, Kleinman D, Chen M, Sordella R, Wilkinson JE, Castillo-Martin M, Cordon-Cardo C, Robinson BD, Trotman LC (2015) MYC Drives Pten/Trp53-Deficient Proliferation and Metastasis due to IL6 Secretion and AKT Suppression via PHLPP2. Cancer Discovery 5: 636-651

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Max Perutz Science Writing Award 2016

I remember, a couple of years ago, seeing an advert by the Medical Research Council (MRC) for a science writing competition and subsequently being bitterly disappointed when I found out it was only for PhD students. Luckily, it’s an annual competition and even more fortunately, The Francis Crick Institute is partly funded by the MRC so that I was eligible to enter.

Now – spoiler alert – before this post ends with an absolute anti-climax, I’ll tell you straight away that I didn’t win. However, I enjoyed answering the question why my research matters in the 800-word essayNot all cancer cells are equal“. The judges used three main criteria to evaluate the essays: 1) Does the essay convincingly explain why the research matters? 2) Is it easy to understand for a public audience? 3) Is the essay well written?

Although I didn’t win, I was shortlisted together with thirteen other entrants and got to attend a science writing masterclass led by Jon Copley, the co-founder of SciConnect, a company that provides science communication training to scientists. The News and Features producer at the MRC was live-tweeting from this course – how cool is that?

The class was really helpful. For instance, I learnt that when writing short to medium length articles (up to 1000 words maximum) the most common structure is the “inverted triangle”. The most important information goes first, i.e. my research matters because it may lead to the development of new anti-cancer drugs. This is different from a research article because there the discussion and conclusion are arguably the most important and come last. I think most essays, including mine, had introductions that were too long. Another handy tip was to think about when/at what age I last shared a class with my target audience. For these essays we could probably assume that interested readers would have had a science education until GCSE level – so we were supposed to write in a way that a fifteen year old might understand.

inverted-triangle

Inverted triangle essay structure for short to medium length articles – copied directly from Wikipedia

When I looked around the room during the writing class – and you might notice it in the photo – I realised that everyone else was probably British and definitely white. At first I was a little bit confused by this since, surely, there is no correlation between skin colour and English writing skills; of last year’s six Man Booker Prize nominees only two were white. But it all made sense when I looked up the MRC’s PhD student funding policy: students need to be eligible to reside in the UK without restrictions and therefore this skews the demographic. [Why higher education in the UK is not more widely accessed is a whole different kettle of fish.]

all-shortlisters

The fourteen shortlisters together with two of the judges, Chris van Tulleken and Donald Brydon, and Robin Perutz, the son of Max Perutz – image copied directly from the MRC website

To round off the day we were all invited to the ceremony at the Royal Institution that evening. In addition to the actual prize-giving, both Donald Drydon, chairman of the MRC, and Robin Perutz, Max Perutz’s son, gave good speeches. The former emphasised that science communication with the public is more important than ever for securing support and funding, since Brexit probably means there will be less money from the government.

Your ability to explain your science allows us, as a country, to carry on being curious. – Donald Brydon

Robin Perutz told a story, also very topical, about how his father and mother met due to an organisation called the Society for the Protection of Science and Learning (SPSL, founded in 1933), which had the mandate of supporting refugee scientists in the UK. Among others, the SPSL helped sixteen future Nobel Prize winners, among which were Max Perutz, Max Born and Hans Krebs. Other prominent academics included Nikolaus Pevsner and Karl Popper. Robin Perutz, currently a professor of inorganic chemistry at the University of York, explained that his lab is taking/has taken in a scientist from Syria who is being funded by the Council for At-Risk Academics (Cara). And it turns out that Cara is none other than SPSL under a new name.

Lastly, we received a copy of The Oxford Book of Modern Science Writing. Who can say no to a book. Overall, from the actual essay writing to the writing class and the ceremony this was an enjoyable experience, which I would highly recommend. Thanks to all the judges and the MRC staff who organised the award. Congratulations to the winners and other almost winners!

modern-science-writing


Now that I think about it, I’ve actually already written a few things relating to Max Perutz, including about his biography, his optimism in research and a symposium in honour of his 100th birthday. It seems I’m quite the fan.

Not all cancer cells are equal

This is the essay I submitted to the Max Perutz Writing Award 2016.

Look at yourself in the nearest mirror and, if you aren’t too squeamish, visualise the inside of your body. It’s obvious that not all your cells are the same. We are made of many different tissues that perform different tasks: skin cells protect us from the environment, white blood cells defend us against infections, nerve cells allow us to move and think. Cancer – the uncontrolled growth of cells – can arise from virtually any type of tissue. We hear about new treatments for skin cancers, about raising money for childhood leukaemias, about inoperable brain tumours. We know that there are different types of cancer.

But an individual tumour in a tissue is also complex. Researchers realised decades ago that, like our healthy bodies, tumours aren’t simply lumps of identical cells; that within each tumour there are different cell types. For instance, some tumour cells divide indefinitely to keep the cancer alive, others invade into surrounding tissue and spread to other sites of the body, while yet others stimulate blood vessels to grow. Some cancer cells even combine several of these properties.

In our laboratory we study the pancreas, an organ of the digestive system, which aids digestion and controls metabolism throughout the body by secreting hormones such as insulin. In particular, we investigate variations among cell types in the most common kind of pancreatic cancer called pancreatic ductal adenocarcinoma (PDAC for short). PDACs are among the most deadly cancers with only about three per cent of patients diagnosed with PDAC in the UK surviving for longer than five years. One of the reasons for this gruelling statistic is that PDACs are often diagnosed late, when the cancer cells have already spread to and wreaked havoc in other internal organs. Previously, several labs, including ours, noticed that some PDAC cells are more aggressive than others, more capable of re-growing new tumours from scratch. Now, we aim to understand what makes the more aggressive PDAC cells different from the rest of the cancer cells and how they contribute to the deadliness of this cancer. With that knowledge in hand, the broader aim will be to find anti-cancer drugs to target and kill the most dangerous cells that lie at the heart of PDAC.

A previous PhD student in our lab discovered that the more aggressive PDAC cells make and display large amounts of a certain protein – let’s call it protein X – on their cell surfaces. We say that the more aggressive cells are “marked” by protein X. This realisation was my gateway into finding out exactly how these two cell types, the more and less aggressive cells, differ.

First, I wanted to know whether protein X not only marks the more aggressive cells but whether it is directly responsible for making those cells more dangerous. Therefore I experimentally reduced or elevated the levels of protein X in PDAC cells we grow in the lab. Then I assessed whether the PDAC cells grew more or fewer, larger or smaller “organoids”, miniature replicas of pancreatic tumours. Astonishingly, the cancer cells actually grew less well when I removed most of protein X, or they divided and proliferated much more when they had more of protein X. This is a good indication that, in future, drugs might be delivered directly to protein X to eliminate the aggressive cells or convert them into tamer cells.

In the meantime, I am on the lookout for other characteristics that might distinguish between the more and less aggressive cells. From one of my experiments I have data hinting that the two cell types might in fact have different physical properties. However, until I’ve repeated these experiments I can’t be certain that this difference in appearance contributes to the more aggressive cells’ behaviour. But it is thinkable, for example, that the more aggressive cells can attach to other cells or blood vessels more easily, aiding their movement to the lungs or liver. These secondary tumours, also known as metastases, are the tumours that PDAC patients usually die from. Next, I need to determine whether there is a direct connection between protein X and the variations among the physical properties of the PDAC cells.

We really want to pin down the differences between the more and less aggressive cells so that hopefully researchers and pharmaceutical companies will be able to design and develop more effective drugs to tackle PDAC. In a few years, once we know more precisely what protein X is doing in the more aggressive cells, our findings might matter a great deal to patients. For the moment I am simply trying to find out more about how PDAC cells work and I know that can sound theoretical. However, I am certain that knowing why and how some cancer cells, clearly, are more equal than others will help patients in the future.

 

A Second Word on Evolution

Life as a PhD student is busy and doesn’t leave much time for other activities, including this blog. So last time, about a month ago, I left you with the question of how the genetic code may have evolved over time.

For decades some scientists have hypothesised that the genetic code evolved by a so-called direct templating mechanism (also known as the stereochemical hypothesis). That is, the strings of ribonucleotides that make up an RNA molecule could physically interact with amino acids, the building blocks of proteins. This interaction would promote the reaction of adjacent amino acids to start forming a longer polypeptide chain. For a review on the different hypotheses see Koonin & Novozhilov (2009).

One of the proponents of the stereochemical hypothesis is Bojan Zagrovic and his research group at the Max F. Perutz Laboratory in Vienna. They have published several papers on this topic and almost a year and a half ago I went to a symposium where Bojan Zagrovic gave a talk on exactly this topic. I wrote about the various presentations I heard there and then several months later a friend I had met during the Cold Spring Harbor Laboratory (CSHL) undergraduate research programme sent me a message saying he had been inspired, by the blog post, to do some research of his own.

In particular, John wanted to investigate whether there was a pattern behind the observed interactions between the amino acids in proteins and the ribonucleotides in RNA. To do this he and Rachel (another student from CSHL) used computational biology approaches to study a large published dataset of protein-RNA complexes. They found that there is a correlation between these physical interactions and the way the genetic code is laid out.

Once these findings had been made they wrote up a draft manuscript, including some figures, which were produced by Grace, a colleague of John’s at Carleton College in the USA. John asked whether I would mind reading the manuscript to give feedback and of course I was happy to do that. We started e-mailing back and forth and decided to extend the computational experiments, and I edited and expanded the text.

The most interesting result was that we could use the knowledge derived solely from the interaction data (blue and red bars) to predict, significantly more accurately than expected by chance (yellow bars), the amino acid sequence of a protein from its mRNA precursor:

Screen Shot 2015-12-07 at 23.00.28

Combining amino acid-nucleobase affinities with mRNA nucleobase content to predict amino acid sequences without universal genetic code. Copied directly from our paper.

In particular, the proteins that form the ribosome – the molecular machine that translates mRNA into protein in modern-day cells – were more accurately predicted than a random protein from our dataset, possibly suggesting that direct interactions between RNA and amino acids led to the formation of the first primitive ribosomes. However, as you can see, the prediction accuracies do not exceed 15% so all results from this paper need to be taken with a pinch of salt; I think the best we can do is say that our results strengthen the stereochemical hypothesis but by no means prove it. [In any case, the scientific method is only good at disproving theories.] Since the journal, Scientific Reports, is an open access journal anyone can read the paper here.

Overall, I am just proud that we managed to publish our work after a long and iterative process, including one revision. All of this was done long-distance via Skype and e-mail. We were all working or studying full-time at the same time and moreover, we did this without the help of a professor/group leader. In fact, none of us even has a PhD (yet).

Lastly, I have noticed a mini-surge in views of my blog posts pertaining to PhD interviews. Clearly the invitations for the next year have been sent out and I hope whoever is reading this is finding it helpful and: good luck!

References:

Cannon JGD, Sherman RM, Wang VMY, Newman GA (2015) Cross-species conservation of complementary amino acid-ribonucleobase interactions and their potential for ribosome-free encoding. Scientific Reports 5: 18054

Hlevnjak M, Zagrovic B (2015) Malleable nature of mRNA-protein compositional complementarity and its functional significance. Nucleic Acids Research 43: 3012-3021

Koonin EV, Novozhilov AS (2009) Origin and evolution of the genetic code: the universal enigma. IUBMB Life 61: 99-111

Polyansky AA, Zagrovic B (2013) Evidence of direct complementary interactions between messenger RNAs and their cognate proteins. Nucleic Acids Research 41: 8434-8443

de Ruiter A, Zagrovic B (2015) Absolute binding-free energies between standard RNA/DNA nucleobases and amino-acid sidechain analogs in different environments. Nucleic Acids Res 43: 708-718

A Word on Evolution

When we think of evolution the first things that come to mind are probably Darwin’s finches, the survival of the fittest and the emergence of ever larger and more complex species that originally derived from single cells. Maybe some people think of how the extinction of dinosaurs allowed mammals to flourish or how the appearance of oxygen-producing cyanobacteria 2.3 billion years ago decimated the number of living species drastically. (According to Wikipedia this event is also known as the Great Oxygenation Event or the Oxygen Holocaust.)

Although I had the opportunity to take a course on “Evolution and Behaviour” during my undergraduate degree, I chose other options instead, including physiology, pathology and (bio)chemistry. Therefore it’s interesting to me how evolution happened on a smaller scale, namely at the level of the molecules that make our cells do what they normally do: DNA – RNA – proteins.

However, cells have not always existed and neither have these molecules. A prominent theory suggests that of those three, RNA evolved first and acted both as an entity that can store information (much like DNA does today) and as a catalyst to drive reactions. This is known as the “RNA world hypothesis” (for a recent review, see Higgs & Lehman, 2015). Although modern cells mainly use proteins to carry out chemical reactions, RNA is still necessary for some very important reactions, including RNA splicing (a necessary part of gene expression in eukaryotes) and protein formation.

The fact that RNA is necessary for the production of proteins from single amino acids strongly supports the idea that RNA preceded proteins during molecular evolution. Today the formation of proteins – also known as protein translation – is carried out by a huge complex, called the ribosome, made of both proteins and RNA:

yeast ribosome

Crystal structure of the S. cerevisiae (yeast) ribosome comprising both RNA and protein – copied directly from Ben-Shem et al., 2010

The ribosome is so large that it proved difficult to study, but when crystal structures finally became available this work was rewarded with the Nobel prize for Chemistry in 2009 (Venkatraman Ramakrishnan, Ada Yonath and Thomas A. Steitz).

Since evolution generally tends from the more simple to the more complex these ribosomes had to gradually come into existence. However, this begs the question how DNA/RNA was translated into proteins before ribosomes? We know now that ribosomes “translate” the so-called genetic code: triplets of RNA bases (adenine A, guanine G, cytosine C and uracil U) get translated into one of twenty amino acids. This code was cracked in the 1960s after the structure of DNA had been solved. There are 64 possible triplets (4x4x4) and only 20 amino acids so that in some cases several triplets encode the same amino acid:

genetic_code_med

Table of the genetic code – copied directly from here.

If one looks at this table a bit more closely it becomes obvious that the code is not random. For example, the amino acid glycine (bottom right) is encoded by four triplets that each start with two guanines, and valine (bottom left) always has G-U at the beginning of its triplets. In theory there are 1.5 x 10^84 possible ways of arranging this table – for comparison, there are approximately 4 x 10^80 atoms in the observable universe – and therefore it is extremely unlikely that the code used today arose by chance.

So how did the genetic code evolve? As in most of science there are several competing, but not necessarily mutually exclusive hypotheses about this. One of them argues that the code arose by natural selection so that mutations at the third base of a triplet would not change the protein sequence. For example, if the DNA sequence GGT were mutated into GGA this would not change the glycine at that position. The code provides robustness to an organism whose DNA is constantly being damaged/mutated. Some scientists therefore propose that the triplet code was originally a quadruplet code – increasing the so-called redundancy – which would have allowed for even more DNA damage without changing the amino acid sequence within a protein.

There are other theories too. Can you think of any? Stay tuned for some exciting news because, yes, there is a good reason that I am writing about this topic and not the more usual CRISPR/cancer.

References:

Ben-Shem A, Jenner L, Yusupova G, Yusupov M (2010) Crystal Structure of the Eukaryotic Ribosome. Science 330: 1203-1209

Higgs PG, Lehman N (2015) The RNA World: molecular cooperation at the origins of life. Nat Rev Genet 16: 7-17