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:

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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:

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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:

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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

Still digesting…

… the outcome of the Brexit referendum, yes. Or the fact that Austrian presidential elections need to be repeated, yes. But also, and on a more positive and scientific note, still digesting articles at eLife. It’s almost exactly a year since I did a short internship with their Features Editorial team, at the end of which my boss, Peter Rodgers, asked whether I would consider continue writing as a freelancer. Consider it? Of course. Yes, no consideration needed. I don’t think I could conceal quite how pleased I was with the offer. So for almost a year now I have been writing one digest a week (about two hours worth of work) and here I’d just like to highlight a few of the most interesting ones.

Inactivation of the ATMIN/ATM pathway protects against glioblastoma formation

This was the second paper that landed in my inbox to digest. When I read the subject line I was a bit baffled by the coincidence, because it surely had to be a coincidence. The lead author of this paper was none other than my current PhD supervisor with whom I was scheduled to start a month later.
The main finding of this paper was a little bit counter-intuitive. The first author, Sophia Blake, studied glioblastomas, the most aggressive form of brain cancer, iand found that when she deleted a tumour suppressor gene called p53 in mice, the animals developed these tumours. So far so good. However, when she deleted a second tumour suppressor called ATMIN at the same time, fewer mice got fewer and smaller tumours.  The paper then goes into some mechanistic detail of how this happens and finishes by showing that there are probably similar processes at play in human glioblastomas.

Ebola virus disease in the Democratic Republic of the Congo, 1976-2014

Most often the papers I read and digest are about cancer, stem cells or molecular biology. Here, however, I got to take a look at an epidemiology study: the authors compiled data for seven Ebola outbreaks in the Democratic Republic of the Congo. To me the most interesting observation was that outbreaks that had, at the outset, a high “reproduction number” – the number of people a single infected person transmits the disease to – were caught and contained early. However, when this reproduction number was smaller than about three the outbreaks seemed to be dealt with less quickly, leading to an overall greater negative effect.

Pericytes are progenitors for coronary artery smooth muscle

In this paper Volz et al. used fluorescent imaging to track the progression of epicardial cells (on the surface of the heart) deep into the muscle tissue of the heart. Using these microscopy techniques, the authors could follow how the epicardial cells become smooth muscle cells, cells that contract and relax, in the coronary arteries. Clicking on the image below will take you to a video consisting of snapshots taken from the outside of a mouse heart to further within. The epicardial cells first become so-called pericytes, cells that normally support blood vessels, and then eventually turn into smooth muscle cells.

pericytes

Snapshot from the first video in Volz et al.

Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties

This last paper I want to mention briefly because it is on a subject that is similar to my project. Fessart et al. studied what can make lung and breast cancer cells more aggressive, more tumorigenic. They noticed that a protein called AGR2, which is normally found within cells where it helps to fold other proteins correctly, can also be secreted outside cells. When this happens AGR2 can make healthy lung cells cancerous.

Almost one year of PhD is already over, three more to go. I think we can count ourselves lucky if, by the end of it, we have a nice story to publish…

References:

Blake SM, Stricker SH, Halavach H, Poetsch AR, Cresswell G, Kelly G, Kanu N, Marino S, Luscombe NM, Pollard SM, Behrens A (2016) Inactivation of the ATMIN/ATM pathway protects against glioblastoma formation. eLife 5: e08711

Fessart D, Domblides C, Avril T, Eriksson LA, Begueret H, Pineau R, Malrieux C, Dugot-Senant N, Lucchesi C, Chevet E, Delom F (2016) Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties. eLife 5: e13887

Rosello A, Mossoko M, Flasche S, Van Hoek AJ, Mbala P, Camacho A, Funk S, Kucharski A, Ilunga BK, Edmunds WJ, Piot P, Baguelin M, Muyembe Tamfum J-J (2015) Ebola virus disease in the Democratic Republic of the Congo, 1976-2014. eLife 4: e09015

Volz KS, Jacobs AH, Chen HI, Poduri A, McKay AS, Riordan DP, Kofler N, Kitajewski J, Weissman I, Red-Horse K (2015) Pericytes are progenitors for coronary artery smooth muscle. eLife 4: e10036

Righting wrongs & reading writings

Now this story is not invented, and reality is always more complex than invention: less kempt, cruder, less rounded out. It rarely lies on one level. – The Periodic Table by Primo Levi

Today I would like to firstly share a book recommendation: The Periodic Table by Primo Levi. Levi, born in Turin, Italy in 1919, was a chemist by training and a writer by necessity. He also happened to be Jewish. The Periodic Table (1975) contains, chapter by chapter – each named after an element that featured in Levi’s life, work or imagination – short stories, memories and anecdotes: ranging from the history and ancestry of his own family, to the story of a lone lead miner, the attempts at finding closure after surviving the concentration camp at Auschwitz, all the way to the life-affirming story of a single atom of carbon. You don’t have to be a chemist (although you can be) to enjoy the humour, the writing style and the humanity of this collection. It would make a good pairing with the psychologist Viktor Frankl’s book Man’s Search for Meaning (1946; the original German title …trotzdem Ja zum Leben sagen more literally means “Yes to Life despite it all”), a different survivor’s account of Auschwitz.

primo-levi

Primo Levi – image copied from Nature


And on another more literary than scientific note: I had the opportunity to attend a talk by Brett Benedetti, an Associate Editor at Nature Medicine. He explained the processes that manuscripts go through when they are received by the journal’s editorial team and what “tricks” authors can use to give their manuscripts a better chance. Among these were so-called “pre-submission enquiries”, which are letters (i.e. e-mails) that potential authors can send in advance of their manuscript to double check whether their work will be appropriate to the journal. In many cases this may save a lot of time if the answer to the enquiry is a resounding “no”: the work can be revised or immediately sent to a different journal instead, without going through the time-consuming process of submitting the entire manuscript.

The most common reason that papers are rejected from leading journals such as Nature, Science or Cell is that they are not “novel” enough. After this talk I think I have understood a little better what exactly is meant by novel: if you imagine knowledge in a scientific field as a two-dimensional line or arrow, it is never a smooth line. There are always gaps and, maybe more often than not, branches that come to a sudden halt. So something that is novel and publishable in high-impact journals contains work that pushes this arrow further and does not merely fill in the preceding gaps. This kind of work, of course, is no less important and needs to be done rigorously, perhaps even more so than those types of experiments that are novel.

Lastly, when asked by the audience about his own career path into scientific editing/publishing, Benedetti replied that he had had no experience in this field whatsoever (apart from the scientific prerequisites of a PhD and post-doc as a neuroscientist). I found this surprising but also highly encouraging.

Editing @eLife

Within the span of three days two people independently told me that I “always manage to land on my feet”. Well, I think they’re right about that. For the last two weeks I have been working as an editorial intern in the Features Team at the scientific journal eLife.

eLife logo

eLife logo (copied directly from their website)

They won’t object to me re-using their logo here since eLife was established in 2012 as one of the first fully open-access online journals for the life and biomedical sciences. One of eLife’s distinguishing features is its fast decision-making process: once a research team submits an article for consideration the initial decision of whether to review it or immediately reject it is made within a few days. Furthermore, the reviewing process itself rarely takes more than a month and if changes need to be made to the manuscript then there usually is only one round of revisions. So when it took them a whole nine days to reject a paper I had been working on with friends they were actually being slow. Other than having a fast decision-making process, eLife is also trying to increase the quality of the science they publish and one endeavour that I find particularly interesting is their “cancer reproducibility project“: 50 of the highest impact cancer papers published between 2010 and 2012 were picked and are now going to be re-done by independent researchers in an attempt to find out how reproducible the results actually are.

In the day-to-day publishing at eLife, however, one of the main things I worked on was writing so-called Digests – short summaries of each of the research papers that are meant to be understandable to interested laypeople. The digests include some background information, an explanation of the main results in the paper and a brief description of which questions future experiments will address. Among other things, I wrote about what happens to sleep-deprived fruit flies, a new mechanism that protects against pancreatic cancer, how some pathogenic gut bacteria get past our defences, and how skin cancer cells move. After reading and thinking about a considerable number of these articles it is less surprising that our paper was rejected.

Of course my digests are not being published as I wrote them. Peter, the main Features editor, went through and corrected all of my writing, something that was extremely useful to me. For instance, I learnt to avoid what he calls “jaw-breakers”, combinations of words in quick succession that are difficult to say. (However, upon re-reading that last sentence, maybe I should say I am still “learning to avoid” jaw-breakers.) Other things he pointed out were practical, because they helped me understand how much or how little the general public can be assumed to know about science. By the start of the second week I could already hear Peter’s voice in my head while writing – which sounds a lot worse than it was since he actually has a pleasant Northern Irish accent – telling me to rephrase this or shorten that.

Apart from writing digests I edited a so-called Insight article, a slightly longer article that comments in more depth on an original research paper and is written by an expert in the field. In particular, I learnt about how the production of transgenic pigs might be able to curb the next outbreak of foot-and-mouth disease (assuming these pigs will be approved by the various regulatory bodies).

Other than that I worked on editing an interview with an early career researcher, i.e. a researcher who is at PhD level or higher but has not received a tenure-track position (yet). In between these things I proofread some articles before they were published in their final form, or looked for “pull quotes” to make articles more interesting. (I didn’t know that pull quotes were called that, but they are the sentences that are pulled out of the text of an article and enlarged so that you immediately read them and subsequently get slightly annoyed when you find them again in the main text.)

the features team

Stuart, Peter, me, Emma and Sarah (from left to right)

I have to admit that working in a typical open-plan office was a new experience for me, since I’m generally used to chaotic lab benches and cramped desk spaces. The thing I enjoyed least was that everyone eats lunch on their own (except for Friday pub lunches) and often at their desk. This struck me as quite strange since you would expect it to be much easier to coordinate having lunch together in a place where everyone works at a computer. In labs people are busy doing experiments but somehow they still find time to be a bit more sociable during their breaks. However, having a nine-to-five (or 9.30 to 4.30 …) job is certainly one of the perks of working in an office like this. Furthermore, I think there is generally less chance of one taking work home (both physically and mentally), although of course I imagine that changes as one assumes more responsibility.

Overall I had an enjoyable experience and am grateful for having been able to get a glimpse into real scientific writing, editing and publishing. If I continue writing eLife digests as a freelancer this will give me the benefit of keeping up to date with the latest, high-quality biomedical research outside the narrow range of a PhD. So really all there is left to say is Thank You to Peter, Emma, Sarah and Stuart, and the rest of the eLife team for being so helpful and welcoming.