Drosophila Tumor Suppressors and Mass Spectrometry

During the past years, new insights into stem cell biology have significantly changed the way we think a tumor arises. While it was previously thought that each cell in a tumor is equal in its potential to maintain tumor growth and initiate new tumors, recent experiments have suggested that only a small fraction of cells is capable of maintaining tumor growth. These cells have stem cell characteristics and are therefore called tumor stem cells. Although the tumor stem cell hypothesis may not be applicable to all tumors, it has profound implications for tumor treatment since only therapies that destroy the tumor stem cells will be successful. It is the aim of this proposal to use the fruitfly Drosophila melanogaster to understand the molecular mechanisms that control proliferation in stem cells and to characterize the defects that turn normal stem cells into tumor initiating cells. We have recently identified the protein Brat as a major regulator of proliferation in Drosophila neural stem cells. During stem cell division, Brat is segregated into only one of the two daughter cells, where it is thought to inhibit cellular growth and proliferation. In brat mutants, all daughter cells proliferate and maintain stem cell characteristics leading to an exponential expansion of the stem cell pool and ultimately to neoplastic tissue overgrowth and the formation of a stem cell derived tumor. Brat is conserved in humans and its homologs are deregulated in human tumors suggesting that they might have a similar function in higher organisms as well. However, the molecular mechanisms through which Brat controls cellular proliferation in stem cells are not understood. We propose to use quantitative mass spectrometry to identify binding partners of Brat by immunoprecipitation. Since Brat has been shown to act as a translational repressor outside the nervous system, we will use differential isotope labelling of wild type and brat mutant cells together with proteome-wide quantitative peptide analysis by mass spectrometry in order to find proteins that are directly or indirectly regulated by Brat. Experiments will be carried out in close collaboration between a Drosophila group and a research group specialized in mass spectrometry. This close collaboration will allow the establishment of state-of-the art chromatography and mass spectrometry methodology and their adaptation to the specific biological problem. The highly developed genetic and cell biological tools available in Drosophila will allow rapid functional characterization of the identified proteins. We will use integrase-mediated generation of overexpression lines to overexpress genes potentially repressed by Brat. For loss-of-function analysis, we will make use of a genome-wide collection of inducible RNAi lines that allows the functional analysis of even a large number of genes by knock- down both in wild type and in brat mutant backgrounds. Together, these experiments will help to understand the molecular mechanism through which Brat controls proliferation in Drosophila neuroblasts and might contribute to our understanding of the molecular defects that turn normal stem cells into tumor initiating cells.

During the past years, new insights into stem cell biology have significantly changed the way we think a tumor arises. While it was previously thought that each cell in a tumor is equal in its potential to maintain tumor growth and initiate new tumors, recent experiments have suggested that only a small fraction of cells is capable of maintaining tumor growth. These cells have stem cell characteristics and are therefore called tumor stem cells. Although the tumor stem cell hypothesis may not be applicable to all tumors, it has profound implications for tumor treatment since only therapies that destroy the tumor stem cells will be successful. It is the aim of this proposal to use the fruitfly Drosophila melanogaster to understand the molecular mechanisms that control proliferation in stem cells and to characterize the defects that turn normal stem cells into tumor initiating cells. We have recently identified the protein Brat as a major regulator of proliferation in Drosophila neural stem cells. During stem cell division, Brat is segregated into only one of the two daughter cells, where it is thought to inhibit cellular growth and proliferation. In brat mutants, all daughter cells proliferate and maintain stem cell characteristics leading to an exponential expansion of the stem cell pool and ultimately to neoplastic tissue overgrowth and the formation of a stem cell derived tumor. Brat is conserved in humans and its homologs are deregulated in human tumors suggesting that they might have a similar function in higher organisms as well. However, the molecular mechanisms through which Brat controls cellular proliferation in stem cells are not understood. We propose to use quantitative mass spectrometry to identify binding partners of Brat by immunoprecipitation. Since Brat has been shown to act as a translational repressor outside the nervous system, we will use differential isotope labelling of wild type and brat mutant cells together with proteome-wide quantitative peptide analysis by mass spectrometry in order to find proteins that are directly or indirectly regulated by Brat. Experiments will be carried out in close collaboration between a Drosophila group and a research group specialized in mass spectrometry. This close collaboration will allow the establishment of state-of-the art chromatography and mass spectrometry methodology and their adaptation to the specific biological problem. The highly developed genetic and cell biological tools available in Drosophila will allow rapid functional characterization of the identified proteins. We will use integrase-mediated generation of overexpression lines to overexpress genes potentially repressed by Brat. For loss-of-function analysis, we will make use of a genome-wide collection of inducible RNAi lines that allows the functional analysis of even a large number of genes by knock-down both in wild type and in brat mutant backgrounds. Together, these experiments will help to understand the molecular mechanism through which Brat controls proliferation in Drosophila neuroblasts and might contribute to our understanding of the molecular defects that turn normal stem cells into tumor initiating cells.