Lab Head Oral PresentationDetails of one or more current research programmes of the intervieweeHello everyone, so I’m going to speak about the different research programmes that Dr Abud has been undertaking in recent years. Dr Abud’s research area is in the study of intestinal stem cells, how they are controlled, how they differentiate, how they build tissues and more importantly how they behave in human disease states and how they contribute to cancer development. In her epithelial regenerative lab, Dr Abud and her team of researchers look at molecules that regulate these intestinal stem cells and study their specific functional pathways in cell proliferation as well as facilitation in cancer formation by obtaining results from knock out/transgenic mouse models as well as patient samples.

Today I am going to specifically talk 2 projects that Dr Abud is currently conducting: firstly, the relationship between Snai1, Wnt Signaling, Stem Cell and Cancer Development; and secondly DNp73 and its role in tumour genesis.

Dr Abud also currently studies the effect of Wnt signaling on the expression of a gene known as Snai1 in intestinal epithelial stem cells and has an article, entitled ‘Wnt Signaling regulates Snai1 expression and Cellular Localisation in the Mouse Intestinal Epithelial Stem Cell Niche’ published based on her findings. I know this picture is looking very familiar to you all but this is basically representing the structure I’m going to use in order to present Dr Abud’s research programs, as well as to demonstrate a relationship between normal embryonic development and cancer development. Basically, a cell needs to acquire most of/all 6 of these hallmarks in order to be a cancer cell. Our second speaker, Luke, has previously outlined the molecular details of the canonical Wnt signaling pathway and the molecules that participate in this pathway, especially APC, which I will mention again later. So, Wnt signaling pathway is a very important mechanism in cell growth and proliferation, and as he already mentioned the relationship between the level Wnt signaling and tumour formation, I’m just going to summarise it straight to the point that too much unregulated Wnt signaling can be the cause of cancer.

So how does Snai1, our protein of interest, fit into the picture? Snai1 is essentially a transcription factor, and as the name suggests, it is a member of the Snail family. Snai1 has been known to be the factor that keeps cells in the stem cell state and maintaining stemness, which indcueds high level of cell proliferation. In order to test what she has hypothesized about the level of expression of Snai1, Dr Abud did a fluorescent stain in a CBC cell (crypt base columnar – intestinal stem cell) with a marked protein LGR5 that is characteristic to all stem cells being stained with a different colour. The result shows as follow. Therefore, we can conclude that Snai1 is found in places where we would expect a strong signal for cell growth, in this case Wnt, to be found also.

In a disease state, Wnt signaling is almost always found in most processes of cancer development. Dr Abud, in her work, has looked at a knock out mouse model of the APC, a well known tumour suppressor gene that plays a part in the process of getting rid of ß catenin in a cell. If you chop out APC, it means that the Wnt signaling pathway is let to continue without stop button, and this leads to an accumulation of ß catenin. The cell is therefore under a state where it’s being told to keep proliferate, and that is how cancer is developed. So as you could see, there is a much higher expression of Snai1 in the polyps, which are tumourous tissues than in a normal colon tissue, in this immunohistochemical stain.

As Luke has talked to you in more detail the concept of EMT (epithelial to mesenchymal transition), I am just simply going to make a link between EMT and metastasis, which is again another hallmark of cancer. During EMT, an epithelial cell at starting point is very polarized in normal cell development, and this makes them sort of ‘want’ to stay in their local tissue and not actively proliferating. However, during EMT, as well as tumour genesis, they can start to lose their polarity and thus start to become more migratory. This is when epithelial cell is becoming mesenchymal cell. After EMT, epithelial cells become more specialised. Snai1 is a factor that regulates this process. However, if we do have a Snai1 overexpression somewhere in the EMT, we can observe almost always expect metastasis to happen, as it promotes the stemness in these cells and keep them in proliferation state.

So the problem with cancer lies in the fact that the switch is always turn on! And that is the link between normal tissue development and in cancer. Proliferation is the key thing in development but too much proliferation that goes unregulated can be the cause of cancer. In her studies, Dr Abud has found out that even though the Wnt signal is not there, Snai1 is still present. This suggests that there are, in fact, many other pathways that regulate this Snai1 expression. A knock out of Snai1 or any other component of other pathways can go many different ways, but they all results in very dramatic phenotype which can be at either end of the spectrum: tumour development or loss of stem cells. Therefore, the level of Wnt signals have to be just right.

As part of her research, Dr Abud has also found out what is known as the threshold of Wnt signaling during head-to-tail embryonic development of a typical embryo. So we have an increasing gradient of Wnt signals as we go from head to tail. There are many different ways that a tissue can respond to Wnt signal, and that can be a combination of both the level of expression of Wnt receptors for particular parts/organs of the body, or the expression of other molecules that either enhance or inhibit Wnt signals.

Recently, Dr Abud has given a presentation at the Monash Comprehensive Cancer Consortium on the topic of ‘Functional studies of the role of DNp73 and stem cells in Cancer’, which was held in November 2013 at the Hemisphere Conference Centre, South Road, Moorabin. P73 is a protein related to p53 protein. It has a very similar, almost identical structure to that of p53, which is a well-known tumour suppressor protein, and thus p73 is also

thought to function as a tumour suppressor and the expression of p73 in cultured cells is also thought to promote a growth arrest and/or apoptosis similarly to p53. P53 is the most commonly mutated gene in all types of cancer, so for example, if a tumour is to be sequenced for p53, we would expect to find some definite sort of mutation on it. However, when we do the same sequencing for p73, fewer mutations can actually be found. To provide an explanation for this, Dr Abud has examined the structure of the p73 protein and discovered that the protein itself was composed of different spliced variants. Dnp73 is one of those variants and it stands for dominant negative p73. A dominant negative form of a protein is one that can inhibit the function of another protein. This Dnp73 is a like a short truncated version of p73, it can potentially form bonds with both the full-length versions of p53 and p73 and inactivate their functions as tumour suppressors. So the study in essence provides evidence that there can be mutations, though not as frequently seen as those on p53, happening on the full-length tumour suppressor p73 protein that can lead to tumour formation, but on the other hand, there is also evidence provided that its truncated version dnp73 can act as an oncoprotein. Hence, the ongoing research that is happening in Dr Abud’s lab at the moment is the study of the expected upregulation of dnp73 and how that has an impact on colorectal cancer growth in patients. By conducting studies on the level of dnp73 overexpression on mice, Dr Abud and her team are currently trying to mimic the effect this has in the intestine and the on the overall results, with a focus on mortality rates. They are currently studying the molecular pathways that are affected with the overexpression of dnp73 in the mouse intestine. This could potentially provide a link between dnp73 oncoprotein activity and tumour development in patients with colorectal cancer.