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

Cancer Discovery

As of March 31st 2015 I am the proud co-author of “Myc drives Pten/p53-deficient proliferation and metastasis due to Il6-secretion and Akt-suppression via Phlpp2” – snappy title, right? – which was published “online first” in the journal Cancer Discovery. The abstract and author manuscript can be found here. The polished article will appear in the May print issue, I believe.

The first author, Dawid G. Nowak, was my supervisor at Cold Spring Harbor Laboratory this summer, where I participated in a ten-week undergraduate research programme. The main aim of the paper was to characterise molecular differences between prostate cancer (PC) cells of the primary tumour (i.e. at the prostate) and metastasised prostate cancer cells. A well-known change that occurs during the progression of PC is the loss of so-called tumour suppressor genes, in particular they are the proteins called PTEN and p53. Tumour suppressor genes are responsible for preventing healthy cells from overproliferating, so when they are lost there are fewer “molecular brakes” in the cell to stop aggressive growth. In addition to losing tumour suppressors, cancer cells also activate so-called oncogenes, and these can be viewed as drivers of growth. So, broadly speaking, when tumour suppressors are lost and oncogenes constitutively switched on a cancer cell is created.

Dawid’s work shows for the first time that as PC cells become metastatic they switch from using one main “driver” oncogene known as Akt to using a different oncogene known as Myc. Firstly, they just made this observation in various tissue samples and biopsies, but then the lab, led by Lloyd Trotman, tried to elucidate the mechanism by which this “oncogene switch” occurs. What Dawid found was that an extracellular signalling factor called IL-6, normally involved in inflammatory processes, activates Myc, which in turn activates a phosphatase – an enzyme that dephosphorylates its targets – called Phlpp2. One of Phlpp2’s targets is Akt, and Akt is inactive when dephosphorylated. Thus, once this process is set into motion Myc rapidly overtakes Akt as the main driving oncogene.

Needless to say, one of the methods employed in the study was the CRISPR knockout approach. The idea of the CRISPR experiment was to show that in a PC cell line derived from a patient’s bone metastasis (PC3), which has already lost the tumour suppressors PTEN and p53, we could knock out Myc and at the same time decrease the levels of Phlpp2. And indeed this is what the following Western blot shows (copied directly from Figure 6 of the paper) – on the left is the control and on the right the CRISPR approach has completely knocked out Myc; PCNA is a marker for cell proliferation and beta-actin is the loading control, which shows that equivalent amounts of total protein are present in both lanes:

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And then in the obverse experiment Myc was overexpressed in these PC3 cells (again the Western blot is copied from Figure 6 and the left is the control, whereas the right is the experiment). When this was achieved the levels of Phlpp2 also increased and furthermore, the levels of phosphorylated/active Akt decreased:

Screen Shot 2015-04-06 at 21.27.01

One could now argue, however, that this was merely a correlation but not a causative effect. So to prove that the proposed signalling – Myc activates Phlpp2, which inactivates Akt – was actually occurring, the last experiment was performed in PC3 cells in which, in addition to PTEN and p53, Phlpp2 had also been deleted. Here the levels of phosphorylated Akt no longer decreased because the responsible phosphatase was not present (again from Figure 6; left: control, right: Myc overexpression):

Screen Shot 2015-04-06 at 21.33.00

All in all quite an elegant story I think. Hopefully the scientific community studying PC will be able to use these results in further experiments. For my part I certainly learnt a great deal from Dawid last summer and enjoyed helping to prepare the manuscript for publication.

Reference:

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

From Bench to Bedside…

… is an evocative phrase that almost has one believe that busy scientists in their immaculate white lab coats, bustling about a clean high-tech lab filled with glass flasks containing brilliantly blue and luminescent yellow solutions, are delivering “magic bullet” drugs directly to their colleagues in the clinic. In my, admittedly limited, experience nothing could be further from the truth. When I tell people that I “did cancer research” this summer, investigating the differences between primary and metastatic prostate cancer (PC) in a lab at Cold Spring Harbor Laboratory, it sounds a lot more glamorous than it is.

There are probably two major factors that contribute to this somewhat idealised notion of how translational science works. Firstly, I would wager that the majority of the general public is not aware of what a long-winded process scientific discovery is. In the cancer research field, for example, basic cellular processes are usually probed and tested in cell culture models to begin with. If these experiments yield interesting results then the next step will involve using an animal model (often mice) to test whether the previously formed hypotheses hold true in vivo. If, either by sheer luck or rational thought, a drug is identified that can interfere with the process under scrutiny then it can be tested pre-clinically. If this is successful – and the definition of “successful” in this case is complex because of the guidelines from the Food & Drug Administration, for example – then maybe the drug can be used in Phase I (out of III) clinical trials. The clinical trials are expensive and can take several years to carry out and evaluate. So in total it usually takes at least a decade (and this is most likely a very optimistic estimate) for some basic discovery to gain clinical relevance. Of course there are exceptions to this, and despite the length of the process it is still certainly worth doing.

Secondly, I think that a lot of researchers investigating some disease or other are often unaware of the clinical/medical/social/personal implications that a disease has. A few days ago a close friend of my mother’s, who was diagnosed with PC earlier this year, asked me whether I could tell him more about the research I had done and more about PC in general. I didn’t know what to say. Presumably he didn’t really want to hear about the molecular pathways (e.g. PTEN/Akt pathway) involved. Nor is it particularly interesting to an Austrian individual what the current incidence and mortality rate statistics are in the USA. Reading about novel therapeutic agents that are just starting to be tested and won’t be ready for widespread use anytime soon isn’t going to be helpful either. I ended up sending him some review papers that focus on the clinical aspects of PC (e.g. Heidenreich et al. (2013)), but essentially I was helpless. Working with and experimenting on a cell line that, once upon a time, came from a bone metastasis of a patient with PC just isn’t the same as being confronted with a whole human being suffering from the disease.

In summary, therefore, I think more communication between both sides, and education on both sides, are needed. Patients lying on the bed or people standing by the bedside (doctors and relatives alike) often don’t know about how research at the bench works, and understandably, knowledge about basic research isn’t necessarily a priority for them at such a difficult time. Conversely, people standing at the bench usually aren’t doctors and don’t know about the disease, except in a defined experimental setting. Even MD/PhD researchers need to go through the laborious process outlined above.

Are there any ways of improving the situation? As a layperson: ask more questions. As a scientist: learn better ways of communicating, and maybe to enrol in a “Bench to Bedside” PhD programme such as the one offered by UCL.

Reference:

Heidenreich A, Pfister D, Merseburger A, Bartsch G (2013) Castration-resistant prostate cancer: where we stand in 2013 and what urologists should know. European urology 64: 260-265