Okay, so we've spoken about aberrant DNA methylation, aberrant histone modifications and even histone mutations, mutations and histone variants, and a little bit about nuclear architecture. And now, we come to consider the noncoding RNAs, this extra arm of the epigenetic machinery. So, noncoding RNAs, exist in many classes, as you remember we've spoken about previously microRNAs are indeed globally misexpressed in cancer, but we don't really consider them to have an epigenetic role because they are involved in post transcriptional silencing. They don't alter the epigenome, itself. As opposed to piRNAs and long noncoding RNAs, which indeed do alter the epigenome. So we're going to be thinking about, first of all, very briefly, about piRNAs, and then about the long noncoding RNA HOTAIR. Just like with histone variants, or histone modifications and nuclear architecture, we don't yet know a huge amount about the role of noncoding RNAs in cancer. But we have some, some preliminary findings which are quite interesting. So, just to remind you about piRNAs because it's been several weeks since we've talked about them. piRNAs are expressed from transposons and they tend to be expressed in a bidirectional manner. They are processed and then they can either result in post-transcriptional gene silencing like micro RNAs can, or in some cases, they can be imported back into the nucleus. And they can result in RNA-directed DNA methylation, and this is when they're having an epigenetic effect. So it's interesting that we know that piRNA expression is deregulated in cancer in general. So, what we want to think about is what's the role of piRNAs, normally, they're silencing repeats. So, do we find aberrant expression of piRNAs because they repeat, tend to be hypomethylated and often active, or is it because there's a relationship between piRNAs, and stem cell biology and cancer. So, many people think that cancer may have some sort of a stem cell origin or at least that cancer cells have a bit of a stem cell like behavior. That is they're very good at renewing themselves and they're don't exhaust prematurely, so they can continue to divide. So, we know piRNAs are expressed in stem cells and they have an important role in stem cells like they do in the germline. So, we don't yet know much about how this works. But it's certainly possible that the, the deregulated piRNA expression has something to do with those the altered repetitive elements or the stem cell behavior of cancer. That's really all we know about piRNAs and cancer at the moment, unfortunately. We know just a little bit more about the long noncoding RNAs. Again, like piRNAs, they're globally deregulated, but what's the consequence of this global deregulation? In most cases, we don't really know what the consequence is, it's just been found that they might be overexpressed or underexpressed in a particular type of cancer. Although, in some cases, we know that this has a particular prognostic outcome. So, it may be associated with poor or favourable prognosis. So, just like I mentioned at the beginning of this, this week, if we don't yet know the molecular mechanism by which something acts, but we still know it has some sort of consequence for the patient. In other words, it's associated with a good prognosis or associated with a poor prognosis, that can still be quite a useful piece of information, while we can go back and try and find out what's happening at the mechanistic level. The example I would like to give you where we know something about the molecular mechanism with the long noncoding RNA that is overexpressed in some cancers is for HOTAIR. So HOTAIR, I mentioned when we spoke about the long noncoding RNAs in Week 2. HOTAIR air is a long noncoding RNA that it acts in trans and so, it's quite unusual in that sense. It appears to act as a scaffold, because it binds to both PRC2, which you'll remember lays down H3K27 methylation, and to LSD1, and LSD1 demethylates H3K4 methylation. So, what you see here is PRC2 is laying down an inactive mark and LSD1 is removing an active mark. So HOTAIR can bring these two complexes together, and therefore, really consolidate silencing at the target genes. So normally, HOTAIR is expressed from the HOXC cluster, but then it acts on the HOXD cluster. This is why it's acting in trans. Okay? So it seems to act like a molecular scaffold acting in trans, but potentially has some sequence specificity because it's still acting on a HOX cluster. What we know about in cancer is that HOTAIR is overexpressed in breast cancer, and it's particularly associated with metastasis. In this case, what seems to happen is, if you overexpress HOTAIR in vitro, then it will re-target PRC2, they didn't test LSD1 unfortunately, but it re-targets PRC2. What ends up happening is that the gene expression in those cells changes and it looks more like when HOTAIR is normally expressed when it would, would happen in embryonic development. So HOTAIR is being expressed in breast, but it's creating a gene expression signature of more like an embryonic type of cell, an embryonic fibroblast in this case. So this suggests that it's not just HOXD which is it a target for HOTAIR, in this instance of breast cancer, but rather other genes, because what you see is that metastasis suppressor genes tend to be silenced. And this might, might be why you have an association with increased metastasis when you have increased HOTAIR expression. So potentially, HOTAIR is binding not just to HOXD, but also these other regions with which we don't know whether or not there is or is not any particular identity between this transcript sequence and this region in these, in these genes. It remains to be determined. So, this is a very interesting finding with relation to breast cancer, but I guess, oftentimes you'll want to know does this really hold true more broadly or is it just some weird thing that happens in one type of cancer? And what's being become clear relatively recently is actually HOTAIR expression is a poor prognostic indicator because of this assocation with metastasis, not just in breast cancer but also in esophageal cancer, colon cancer and liver cancer. And so, this mechanism needs to be established. How is it that HOTAIR is being targeted to these other regions in the context of cancer? So, what can we do with this knowledge for HOTAIR, or indeed, for any other long noncoding RNA? Well, if we just note that it happens to be associated with a particular prognostic outcome, but we have no real measure of why that is, or we don't understand yet the molecular mechanism. We could still use the expression of long noncoding RNAs, whether they be aberrant overexpression or aberrant decreased expression diagnostically. So you can detect long noncoding RNAs in in samples if you need to. And actually, there's already one that's being used diagnostically, and that is the expression of PCA3. So, this PCA3 is indicative of prostate cancer, but you can detect PCA3 in the urine. And this is really good because it's a non-invasive test, it's not even as invasive as a blood test. It's certainly not nearly as invasive as having to have a biopsy taken of the prostate itself. So this is a useful diagnostic test that uses this long noncoding RNA and here's a picture of this PCA3 long noncoding RNA. Long noncoding RNAs always have this quite elaborate secondary structure and it's thought that this is how they might act as some sort of a molecular scaffold because they have so many binding surfaces a bit like a protein might. But what about therapeutically? If we know, HOTAIR appears to have an poor prognostic outcome and maybe mechanistically because it's re-targeting epigenetic modifier complexes. What could we do about this at the therapeutic level? Well, we actually know that you can knock down long noncoding RNAs. In other words, you can reduce their expression with particular other RNAs. And so, potentially, these could be used in vivo for therapy if it can be worked out exactly to deliver it to the right cells or to make sure that we can get it into the cells effectively. And this is something that's being looked at by pharmaceutical companies at the moment. So, now, we've spoken about all the different types of epigenetic modifications we know about and how they go awry in cancer. So to summarize what we've said so far then, in terms of DNA methylation, we see genome-wide hypomethylation. This is predominantly at the repeat, or at the intergenic intervals. We see tumour suppressor gene hypermethylation. In terms of histone modifications, there are particular genome-wide alterations, you see. But you see also gain of silencing marks and loss of activation marks at the same CPG islands that are methylated. We know there are actually long range alterations to epigenetic controls. There are whole regions, megabase regions, that will be silenced or activated in the context of cancer. And wee know that there's aberrant nuclear architecture and altered expression of the long noncoding RNAs. So altogether, this means that essentially, every level of epigenetic control that we've spoken about over the past five and half weeks now has gone wrong in cancer. So in the next lecture, I'd like to start thinking about, although we don't really have an answer to it yet, how is it that all of these epigenetic aberrations come about?
