What will you be working on at IFOM?
Basically I am going to set up a laboratory working on the molecular aspects of how cells maintain a stable karyotype. This is a particularly important topic because cancer cells do not know how to maintain their DNA. This is one of the most differentiating features of a cancer cell. So if you were to pick a difference between normal cells and cancer cells the first thing that comes to every cancer scientist’s mind is the fact that if you look at the chromosomes they look different.
What causes the instability and what can be done?
What causes instability is a fundamental question which we are trying to address. The major cause is the failure of a surveillance mechanism that overlooks the stability of the DNA. What happens is that when this mechanism, for whatever reason, does not work properly then chromosomes lose their structure and the number of chromosomes is not controlled properly during each cell cycle. So what is the first event that brings about this phenotype is not clear at all and that’s what we are here for. So we are trying to establish a molecular mechanism which underlies the generation of this phenotype. For me the secret lies in the genes that repair the DNA. So we basically are trying to address the function of these genes.
So why are we using the frog model system? Because many of these genes are essential for life so what happens is that the moment that you inactivate the function of any of these genes the cell dies. So there is no valid model system that allows the dissection of the molecular function of these genes. This is particularly important because I would say that 40-50% of human genes are essential for life and what happens is that in order to study a gene, for example, you try to inactivate the gene function and look at what happens to the cell when the gene is not present. By looking at the cell then you try to infer the function of the gene. So this is when you’re lucky but if your gene is essential the only thing that you will see is that your cell will die and also when you try to inactivate the gene in the entire animal, for example in the mouse, the mouse will not survive.
So what happens in the frog? So we use basically an extract of very big cells which are produced by the frog. The frog is a vertebrate, all the frog genes basically are very similar to human genes. So, for example, if you take the most studied tumour suppressor gene, for example like BRCA2, BRCA2 is very conserved in the frog and BRCA2 is a breast cancer and ovarian cancer tumour suppressor gene. So what we do is we make an extract of the frog egg, so we collect the eggs so we don’t harm the animal, we just collect the eggs which are produced spontaneously by the frog. We make an extract and in this extract we have basically all the genes expressed as proteins and now we have access to a large amount of material. What is really useful about this system is that this extract recapitulates in vitro the cell cycle events typical of an embryonic cell cycle. So when the egg is fertilised normally you would have an embryo which rapidly divides to reach the formation of an adult organism. So what we do with this extract is that this extract biochemically reproduces the same reactions that take place in a dividing cell. So we take advantage of this by depleting the proteins which are already there, so these are the maternal proteins, and in this case, for example, we have a maternal BRCA2 gene which we deplete with specific antibodies and then we see what happens to the cell cycle. For example, we see what happens to the DNA replication, to the mitosis, to the segregation. So if any of these essential processes are affected then we can say that BRCA2 genes are required for any of these specific cell cycle steps. This is something which is very difficult to study in any other system because the moment you inactivate BRCA2 you have a dead cell so where do you go from there?
Do you see therapeutic interventions down the line?
What happens is that, for example, the PARP inhibitor is a very appropriate example. So the PARP inhibitor has been found as a drug which selectively kills the cells which are BRCA2 deficient. It’s supposed to work very well in tumours that arise in BRCA2 deficient patients. But what is the molecular mechanism underlying this drug? What we are doing, we are depleting BRCA2 and we’re putting the PARP inhibitor in our extract and then we are recovering the DNA and we are studying the structure of the DNA. So of course you cannot do it in dead cells but now instead the extract doesn’t die, we recover the DNA and we look at the DNA by electromicroscopy which is an amazingly powerful tool to define the details of the chromosome structure. For example, we recently published evidence that the PARP inhibitor is required to prevent a particularly dangerous DNA structure which is called the reverse fork. A reverse fork is a structure which is formed when the cell replication machinery stops for whatever reasons and this induces the formation of a very dangerous DNA appendix which are basically prone to recombination events. So basically this structure can recombine and can start those translocation and transformation events that are common in the cancer in an unstable cancer genome. This means that these events are probably the very first event which leads to the formation of an unstable chromosome. We think that by using this system we have pinpointed the very first event leading to a genome instability.
