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

On the #IceBucketChallenge

I was challenged to do the ALS (amyotrophic lateral sclerosis) ice bucket challenge, but not in the conventional way. The friend who nominated two other people and me graciously gave us 674968 hours to “NOT do a video (though you can if you have the unstoppable urge to throw water on your head) and instead to donate to a charity of your choice”. He is an enigmatic person full of whimsy and paradoxes with a hint of maniacal genius. On top of upturning a bucket of ice cold water over his head in a completely unfazed manner as a sort of side note during his video, he improvised on his violin for two minutes, gracing us with his musical talent. See for yourself:

The whole #IceBucketChallenge makes me slightly nervous, I must admit. On the one hand, it is an admirable thing to raise awareness for motor neurone diseases (MNDs), of which ALS is one, and to prompt people to donate to charities such as the MND Association (primarily based in England) and the ALS Association (USA). However, I wonder how much the people doing these challenges actually know about the diseases: their symptoms, causes and treatments. As far as I can discern from reading the charity websites a lot of the donated money goes into improving patient care, and although this is unarguably the most urgent frontier to which the funds should be allocated, it is also important to think about basic research into ALS. For example, I doubt that most people who did do the challenge (and may or may not have donated) know that experimenting with the brains of fruit flies can lead to new insights into disease pathogenesis (the development of disease), aetiology (the cause of disease) and possibly even reveal a long sought-after diagnostic marker for ALS.

At this point I ought to admit that I also did not know that basic ALS research can be done in Drosophila melanogaster, but my summer internship at Cold Spring Harbor Laboratory (CSHL) was enlightening in this respect. For example, the lab of Joshua Dubnau (http://dubnaulab.cshl.edu/research_projects/neurodegeneration.html) uses fruit flies (and other models) to investigate the role of the protein TDP-43 in ALS and related frontotemporal lobe degeneration, whose gene is mutated in both familial and sporadic versions of the diseases (for a recent review see Robberecht and Philips (2013) Nature Reviews Neuroscience). Although I am not trying to advocate that we donate to CSHL instead, I think it’s important to be aware that basic (animal) research, which, on the face of it, may not seem important to members of the lay public can very often be justified because it will benefit people in the long run.

Lastly, the ice bucket challenge seems wasteful since a large proportion of the world’s population does not have access to clean water. So when Matt Damon was challenged by Ben Affleck and Jimmy Kimmel he decided to use water from the toilets in his house, commenting that that water was still relatively clean compared to what some people have to drink (http://time.com/3181818/als-ice-bucket-challenge-matt-damon-uses-toilet-water/). For others, such as Stephen Hawking who suffers from an MND similar to ALS, pouring ice cold water over one’s head is not exactly conducive to good health. To circumvent this problem the physicist was able to convince his children to do the challenge for him and subsequently nominated the director of the Science Museum in London and the Chancellor and Vice-Chancellor of the University of Cambridge (http://www.independent.co.uk/news/people/stephen-hawking-mnd-sufferer-does-ice-bucket-challenge-with-a-twist-9698539.html).

ALS, also known as Lou Gehrig’s disease, is a rare yet devastating disease, which progresses from muscle weakness and atrophy to difficulties speaking and breathing, and eventually to death. There is currently no cure for ALS, only treatments that improve the quality of life for patients, and therefore knowledge of the exact disease mechanism(s) is vital because only then can drugs be more rationally designed.

Now I will go and donate to Forum ALS, the Austrian equivalent of the ALS Association: a recent news article (http://derstandard.at/2000004617183/Amyotrophe-Lateralsklerose-Etwa-800-Betroffene-in-Oesterreich) seems to indicate that this organisation has been receiving more interview enquiries but not more donations since the ice bucket challenge went viral.