We have a new paper out! This post is to explain what it’s about.
Cancer cells often have gene fusions. This happens because the DNA in cancer cells is really messed up. Sometimes, chromosomes can break and get reattached to a different one in a strange way. This means you get a fusion between one gene and another which makes a new gene, called a gene fusion. There are famous fusions that are known to cause cancer, such as the Philadelphia chromosome in chronic myelogenous leukaemia. This rearrangement of chromosomes 9 and 22 result in a fusion called BCR-ABL. There are lots of different gene fusions and a few years ago, a new fusion was discovered in bladder and brain cancers, called FGFR3-TACC3.
Genes encode proteins and proteins do jobs in cells. So the question is: how are the proteins from gene fusions different to their normal versions, and how do they cause cancer? Many of the gene fusions that scientists have found result in a protein that continues to send a signal to the cell when it shouldn’t. It’s thought that this transforms the cell to divide uncontrollably. FGFR3-TACC3 is no different. FGFR3 can send signals and the TACC3 part probably makes it do this uncontrollably. But, what about the TACC3 part? Does that do anything, or is this all about FGFR3 going wrong?
What is TACC3?
TACC3, or transforming acidic coiled-coil protein 3 to give it its full name, is a protein important for cell division. It helps to share the chromosomes to the two daughter cells when a cell divides. Chromosomes are shared out by a machine built inside the cell called the mitotic spindle. This is made up of tiny threads called microtubules. TACC3 stabilises these microtubules and adds strength to this machine.
We wondered if cancer cells with FGFR3-TACC3 had problems in cell division. If they did, this might be because the TACC3 part of FGFR3-TACC3 is changed.
We weren’t the first people to have this idea. The scientists that found the gene fusion suggested that FGFR3-TACC3 might bind to the mitotic spindle but not be able to work properly. We decided to take a closer look…
What did you find?
First of all FGFR3-TACC3 is not actually bound to the mitotic spindle. It is at the cells membrane and in small vesicles in the cell. So if it is not part of the mitotic spindle, how can it affect cell division? One unusual thing about TACC3 is that it is a dimer, meaning two TACC3s are stuck together. Stranger than that, these dimers can stick to more dimers and multimerise into a much bigger protein. When we looked at the normal TACC3 in the cell we noticed that the amount bound to the spindle had decreased. We wondered whether the FGFR3-TACC3 was hoovering the normal TACC3 off the spindle, preventing normal cell division.
We made the cancer cells express a bit more normal TACC3 and this rescued the faulty division. We also got rid of the FGFR3-TACC3 fusion, and that also put things back to normal. Finally, we made a fake FGFR3-TACC3 which had a dummy part in place of FGFR3 and this was just as good at hoovering up normal TACC3 and causing cell division problems. So our idea seemed to be right!
What does this mean for cancer?
This project was to look at what is going on inside cancer cells and it is a long way from any cancer treatments. Drug companies can develop chemicals which stop cell signalling from fusions, these could work as anti-cancer agents. In the case of FGFR3-TACC3, what we are saying is: even if you stop the signalling there will still be cell division problems in the cancer cells. So an ideal treatment might be to block TACC3 interactions as well as stopping signalling. This is very difficult to do and is far in the future. Doing work like this is important to understand all the possible ways to tackle a specific cancer and to find any problems with potential treatments.
Sourav Sarkar did virtually all the work for this paper and he is first author. Sourav left the lab before we managed to submit this paper and so the revision experiments requested by the peer reviewers were done by Ellis Ryan.
Why didn’t we post this paper as a preprint?
My group have generally been posting our new manuscripts as preprints while they undergo peer review, but we didn’t post this one. I was reluctant because many cancer journals at the time of submission did not allow preprints. This has changed a bit in the last few months, but back in February several key cancer journals did not accept papers that had appeared first as preprints.
The title of the post comes from “Fusion Confusion” 4th track on the Hazy EP by Dr Phibes & The House of Wax Equations.
We have a new paper out! You can access it here.
This paper really was a team effort. Faye Nixon and Tom Honnor are joint-first authors. Faye did most of the experimental work in the final months of her PhD and Tom came up with the idea for the mathematical modelling and helped to rewrite our analysis method in R. Other people helped in lots of ways. George did extra segmentation, rendering and movie making. Nick helped during the revisions of the paper. Ali helped to image samples… the list is quite long.
The paper in a nutshell
We used a 3D imaging technique called SBF-SEM to see microtubules in dividing cells, then used computers to describe their organisation.
Serial block face scanning electron microscopy. This method allows us to take an image of a cell and then remove a tiny slice, take another image and so on. We then have a pile of images which covers the entire cell. Next we need to put them back together and make some sense of them.
How do you do that?
We use a computer to track where all the microtubules are in the cell. In dividing cells – in mitosis – the microtubules are in the form of a mitotic spindle. This is a machine that the cell builds to share the chromosomes to the two new cells. It’s very important that this process goes right. If it fails, mistakes can lead to diseases such as cancer. Before we started, it wasn’t known whether SBF-SEM had the power to see microtubules, but we show in this paper that it is possible.
We can see lots of other cool things inside the cell too like chromosomes, kinetochores, mitochondria, membranes. We made many interesting observations in the paper, although the focus was on the microtubules.
So you can see all the microtubules, what’s interesting about that?
The interesting thing is that our resolution is really good, and is at a large scale. This means we can determine the direction of all the microtubules in the spindle and use this for understanding how well the microtubules are organised. Previous work had suggested that proteins whose expression is altered in cancer cause changes in the organisation of spindle microtubules. Our computational methods allowed us to test these ideas for the first time.
Resolution at a large scale, what does that mean?
The spindle is made of thousands of microtubules. With a normal light microscope, we can see the spindle but we can’t tell individual microtubules apart. There are improvements in light microscopy (called super-resolution) but even with those improvements, right in the body of the spindle it is still not possible to resolve individual microtubules. SBF-SEM can do this. It doesn’t have the best resolution available though. A method called Electron Tomography has much higher resolution. However, to image microtubules at this large scale (meaning for one whole spindle), it would take months or years of effort! SBF-SEM takes a few hours. Our resolution is better than light microscopy, worse than electron tomography, but because we can see the whole spindle and image more samples, it has huge benefits.
What mathematical modelling did you do?
Cells are beautiful things but they are far from perfect. The microtubules in a mitotic spindle follow a pattern, but don’t do so exactly. So what we did was to create a “virtual spindle” where each microtubule had been made perfect. It was a bit like “photoshopping” the cell. Instead of straightening the noses of actresses, we corrected the path of every microtubule. How much photoshopping was needed told us how imperfect the microtubule’s direction was. This measure – which was a readout of microtubule “wonkiness” – could be done on thousands of microtubules and tell us whether cancer-associated proteins really cause the microtubules to lose organisation.
The publication process
The paper is published in Journal of Cell Science and it was a great experience. Last November, we put up a preprint on this work and left it up for a few weeks. We got some great feedback and modified the paper a bit before submitting it to a journal. One reviewer gave us a long list of useful comments that we needed to address. However, the other two reviewers didn’t think our paper was a big enough breakthrough for that journal. Our paper was rejected*. This can happen sometimes and it is frustrating as an author because it is difficult for anybody to judge which papers will go on to make an impact and which ones won’t. One of the two reviewers thought that because the resolution of SBF-SEM is lower than electron tomography, our paper was not good enough. The other one thought that because SBF-SEM will not surpass light microscopy as an imaging method (really!**) and because EM cannot be done live (the cells have to be fixed), it was not enough of a breakthrough. As I explained above, the power is that SBF-SEM is between these two methods. Somehow, the referees weren’t convinced. We did some more work, revised the paper, and sent it to J Cell Sci.
J Cell Sci is a great journal which is published by Company of Biologists, a not-for-profit organisation who put a lot of money back into cell biology in the UK. They are preprint friendly, they allow the submission of papers in any format, and most importantly, they have a fast-track*** option. This allowed me to send on the reviews we had and including our response to them. They sent the paper back to the reviewer who had a list of useful comments and they were happy with the changes we made. It was accepted just 18 days after we sent it in and it was online 8 days later. I’m really pleased with the whole publishing experience with J Cell Sci.
* I’m writing about this because we all have papers rejected. There’s no shame in that at all. Moreover, it’s obvious from the dates on the preprint and on the JCS paper that our manuscript was rejected from another journal first.
** Anyone who knows something about microscopy will find this amusing and/or ridiculous.
*** Fast-track is offered by lots of journals nowadays. It allows authors to send in a paper that has been reviewed elsewhere with the peer review file. How the paper has been revised in light of those comments is assessed by at the Editor and one peer reviewer.
Parallel lines is of course the title of the seminal Blondie LP. I have used this title before for a blog post, but it matches the topic so well.