From stem cell to brain tumor: a genetic analysis

Stem cells have a unique property: They can generate daughter cells that remain stem cells and undergo multiple rounds of self-renewal. At the same time, they give rise to more specialized cell types that ultimately replace cells in the target tissue. While this property makes stem cells an ideal source of cells for regenerative medicine, it also means that the balance between those cell types needs to be precisely controlled. If stem cells are defective and generate only daughter cells, this leads to uncontrolled cellular amplification and it has been proposed that defects like this can contribute to the formation of certain tumors. This project uses the fruitfly Drosophila to understand the mechanisms that control this balance in stem cells of the brain. We focus on the so-called type II neuroblasts, neural stem cells that divide asymmetrically into one cell that remains a neuroblast while the other becomes an intermediate neural progenitor (INP). The INP again divides asymmetrically but now, one cell becomes a ganglion mother cell (GMC) which divides once more into two differentiating neurons. Our project will use an RNAi approach to identify the genes that control the multiple differentiation steps that occur in this lineage. For this, we make use of the fact that defects in lineage progression lead to the formation of a brain tumor. In wild type adult flies, all neural stem cells have differentiated into neurons. In tumor bearing flies, however, the brain is full of proliferating stem cells. We make use of this and generate transgenic flies expressing firefly luciferase under a stem cell specific promoter. This allows us to measure tumor formation simply by placing homogenized fly heads into a luminometer - a very fast and reliable assay. In this background, we will knock down over 80% of all fly genes using the RNAi library established and maintained at the Vienna Drosophila RNAi center (VDRC). Identified tumor suppressors will then be characterized further using cell biological and genetic techniques. Ultimately, we will identify the vertebrate homologs and inhibit them in the mouse brain using "in utero electroporation", a technique that allows for a fast transient introduction of DNA into developing brain cells. Using this technique, we had previously shown a striking functional conservation Drosophila Brat, a key regulator of stem cell proliferation and its mouse homolog TRIM32, which controls neural stem cells in the mammalian brain. We expect that these experiments will lead to new insights into how stem cells control lineage progression and how a stem cell turns into a tumor stem cell to initiate the formation of a deadly brain tumor.

The recent years have seen a major change in our view of tumor development. While it was traditionally thought that tumors are assemblies of cells that have lost the ability to control growth and division but otherwise do not show any cellular hierarchies, it is now becoming increasingly clear that at least some tumors contain stem cells just like normal organs do. In this so-called tumor stem cell hypothesis, all cells in a tumor arise from those stem cells in a cellular hierarchy that is similar to many of the organs in a healthy body. This has strong implications for tumor therapy, as any successful cancer therapy needs to target those tumor stem cells to avoid that they regenerate the tumor mass after successful surgery. The experiments carried out under this grant in collaboration with the laboratory of Prof. Heinrich Reichert (University of Basel) use the fruit fly Drosophila melanogaster as a model system to analyze tumor stem cells, how they arise and what makes them different from normal stem cells. In fruit flies, all neurons in the adult brain arise from stem cells called neuroblasts. These neuroblasts generate a precisely defined number of neurons, but also more stem cells to maintain the stem cell pool. When the balance between those cell types is disrupted, brain tumors arise that resemble human cancer in a striking manner. In particular, pieces of those tumors can be transplanted into another fly where they give rise to another tumor that will kill the host. The genetic tools available in fruit flies have allowed us to narrow down mechanisms responsible for this immortalization.As part of this project, we have first developed tools to obtain pure neuroblasts from healthy and tumerous flies. Using this newly developed technology we could obtain a complete list of all the genes that are turned on or off when neuroblasts become neurons. We were able to assemble a transcriptional network that allows neuroblasts to make identical copies of themselves and identify key components of this network. This network needs to be turned off when neuroblasts become neurons failure to do so results in tumor formation. We could also characterize a protein complex called the SWI/SNF complex as a key regulator of Drosophila stem cells. When the function of this complex is impaired, the daughter cells of neuroblasts that would normally form neurons revert back to additional neuroblasts. This leads to the expansion of the neuroblast pool and ultimately to the formation of brain tumors. The SWI/SNF complex exists in humans as well and recent experiments have shown that it is the most commonly mutated protein complex in human cancer. Thus, our experiments could contribute to the understanding of one of the most important human tumor suppressors.