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

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In silico versus in vitro

For the past five days at least I have felt bad for not updating my blog with some fact about CRISPR or the latest controversy concerning “cancer stem cells”. The reason for this involuntary hiatus is, of course, lab work. All the lab work. All the time. How should I put this; there are distinct benefits to working in silico: leaving the lab/office whenever is convenient, being able to continue work at home, and actually getting home on time for dinner.

Here I am, genuinely happy to be doing work in cell culture (isn’t the dark blue of that lab coat excellent?):

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And then running samples in pre-cast 20-well gels ready to do a Western blot after having treated melanoma cells with a plethora of small molecule inhibitors:

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But then disaster had to strike. It was all going too well. Due to the bubbles rising from the electrodes in the above picture I didn’t have a clear view of the second gel running behind it. What a disappointment:

image2The sad smiley face accurately represents my emotions concerning this gel. Well, at least I know what I’m doing tomorrow.

So I’ll end with a mini CRISPR update: Tsai et al. (2014) developed a new method last year to reduce non-specific cleavage of DNA during genome-editing. They require expression of RNA-guided FokI nucleases, which also cleave DNA, but are only active when dimeric. Each single FokI molecule is guided to its target by a guide RNA, but only when two guide RNAs each bring a FokI to the desired locus do the enzymes become active. This drastically reduces off-target effects because both the sequence and spacing has to be correct. The original CRISPR/Cas9 system has considerable off-target effects, as shown by Lin et al. (2014), for example.

Reference:

Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research 42: 7473-7485

Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotech 32: 569-576

Departmental Research Day & M. Sci. Symposium

The beginning of Lent term was far from gentle. For three days I have been sitting in lecture theatres and seminar rooms. Firstly, for a full day we listened to several professors/group leaders of the biochemistry department describing their research. And secondly, we had two days of the so-called Part III symposium, that is a twenty-minute research update from each of the 31 biochemistry M. Sci. students in the department. (Since then I have started working in a lab again and I have to admit I had forgotten how strenuous it can be.)

First things first. The “departmental research day” was hosted at Robinson College, because the lecture theatre within the department is actually too small to seat all the members of staff and students. The introduction was given by Chris Smith and his most interesting point was probably that the department received an Athena SWAN bronze award last year, which “recognises and celebrates good practice in recruiting, retaining and promoting women in Science, Technology, Engineering, Mathematics and Medicine (STEMM) within Higher Education”. So three cheers for the department!

AthenaSWANBronezAward

The actual research talks by the various professors ranged from mildly piquing to downright riveting. There were several talks on cancer (the head of the department, Gerard Evan, is a cancer biologist so this is hardly surprising): at one end for example, Helen Mott explained how basic biology, crystallography and peptide chemistry are being exploited to research a new class of drugs based on alpha-helical peptides, which are meant to block activity of some small GTPases (sometimes known as cellular switches because they can turn signalling pathways on and off). At the more clinical end, Kevin Brindle demonstrated how techniques such as dynamic nuclear polarisation magnetic resonance imaging (MRI) are progressing to better image biology/cancer in (live) patients. However, the department is also strong in the field of structural biology, since the crystallographer Tom Blundell used to be the head of the department. Furthermore, there is an increasing number of lab groups working on single-celled eukaryotes such as trypanosomes and Toxoplasma.

Additionally, there were at least two overt political references to keep us on our toes. The first one was this:

Screen Shot 2015-01-14 at 20.14.58And I have to say that I wholeheartedly agree. Perhaps unsurprisingly, the professor who used this image in her slides is originally from the Czech Republic and probably quite vehemently opposes the idea of having an in/out referendum in the UK. [Eukaryotes, by the way, are organisms whose cells contain a nucleus and would include plants, animals and fungi, but also single-celled eukaryotes such as trypanosomes and Toxoplasma.]

The second political reference was a quote by Donald Rumsfeld: “As we know, there are known knowns; there are things we know we know. We also know there are known unknowns; that is to say we know there are some things we do not know. But there are also unknown unknowns – the ones we don’t know we don’t know.” He said this in response to questions about Iraq’s involvement in the supply of weapons to terrorist groups. In the context of science this was a comment on the inherent difficulties of modelling biological processes: Steve Oliver uses yeast as a model organism to study metabolic pathways, and in contrast to the qualitative modelling I have been doing, his models are quantitative (i.e. use differential equations and enzyme kinetic data). Interestingly however, both types of model can suffer from similar problems; for example they can be plain wrong, or incomplete, or based on faulty assumptions. And sometimes when we know they are wrong that doesn’t mean we know how to improve them. Not knowing that they are wrong/incomplete (i.e. the unknown unknown) is arguably the most comfortable position to be in.

The following two days were filled with project reports of all the biochemistry M. Sci. students. It is worth noting that several of these talks were possibly more interesting and of better quality than some of those given by the professors. There was an extremely wide variety of topics including: cancer research, developmental biology, disease biology (including the rare lysosomal storage disease called Krabbe disease), in vitro enzyme evolution, structural biology (including taking trips to the x-ray source near Oxford, the Diamond Light Source), stem cell biology and research into the origins of life. This latter research project is investigating how the first RNA molecules may have come together to form larger, catalytic molecules of RNA (“RNA world hypothesis”), and to do this the reactions are carried out at -9ºC in the eutectic phase of water-ice, a condition thought to mimic prebiotic chemistry.

Lastly, what would a blog post be without the mention of CRISPR. At least two of the M. Sci. projects involve the use of this genome-editing technology. In one case it will be used to knock-out a microRNA that may be involved in the regulation of bicoid mRNA during Drosophila (fruit fly) development. And in the other case it is being used to target a transcription factor that is implicated in the regulation of stem cell fate. Interestingly, the strategy here involves using two guide RNAs simultaneously, both targeted to within the gene of interest, with the aim of creating a large deletion rather than just a small insertion/deletion.

Needless to say, the progress of all our projects is far slower than we (and probably our supervisors) would have hoped.

Project Update #2

After struggling slightly with the immensity of the task at hand, we (my supervisor(s) and I) have decided to delineate the project slightly more clearly: instead of studying important cancer pathways as they occur in fibroblasts (cells that mainly serve structural purposes in the body and are involved in wound healing), the research will now focus on modelling pathways involved in skin cancer. In particular, the evolution of skin cancer from initial benign states to primary and subsequently metastatic disease (melanomagenesis) will be modelled. Hopefully the work will provide some insight into how melanoma could be treated more effectively.

At the moment, although effective therapies do exist to treat melanoma, the patients almost inevitably relapse after a while, due to resistance to the drugs. The following images (from Wagle et al. (2011)) are not for the faint-hearted. This patient had advanced metastatic melanoma:

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He was treated with a drug that inhibits one of the main “drivers” of the tumours (a protein kinase, which is constitutively activated), and the nodules entirely disappeared:

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However, a couple of months later the tumours re-emerged and this time they were resistant to the drug. The man died a few weeks later.

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A better understanding of the pathways involved in melanomagenesis and drug resistance mechanisms may be able to inform future therapy options. Better treatment may mean that we can pre-empt these relapses and prevent them from occurring.

Reference:

Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, Pochanard P, Kehoe SM, Johannessen CM, MacConaill LE, Hahn WC, Meyerson M, Garraway LA (2011) Dissecting Therapeutic Resistance to RAF Inhibition in Melanoma by Tumor Genomic Profiling. Journal of Clinical Oncology 29: 3085-3096

Project Update #1

I’ve been back in Cambridge for four days and have already met and talked to at least four different people involved in supervising and assisting with my master’s thesis project.

Luckily, this means that I now have a somewhat clearer idea of what I’ll be doing to begin with. In essence, I think, the whole project – my project is a sub-project of the PhD student’s project in Jasmin’s lab – is an admirable undertaking. The general plan is to recreate gene and protein regulatory networks (focussing on major players in cancer) in silico by sifting through the vast amounts of published data. This should yield a simplified but useful model, which can subsequently be tested by inputting experimental data that was not used to build the model. If this works then the model can be asked to predict new outcomes when parameters are changed. Any interesting results from these simulations can then be tested in the lab, but currently that is still quite far in the future.

To begin with I will be doing a lot of literature review and model building, the prospect of which is actually very exciting because I enjoy reading papers and trying to organise data into more accessible (graphical) models.

P.S.: While I wrote this short entry the 2014 Nobel prize in Chemistry was announced. The winners are Eric Betzig, Stefan Hell and William Moerner “for the development of super-resolved fluorescence microscopy”. This means that a) both the Physics and Chemistry prizes this year were awarded for very practical, technological advances, which incidentally are both to do with visible light, and b) the Chemistry prize was once again very biological. Clearly I’m not complaining and I agree with the committee’s announcement this morning, “Biology has turned to chemistry. Chemistry has turned into biology.”