ABSTRACT
Metabolic reprogramming is a key feature of many cancers, but how and when it contributes to tumorigenesis remains unclear. Here we demonstrate that metabolic reprogramming induced by mitochondrial fusion can be rate-limiting for immortalization of tumor-initiating cells (TICs) and trigger their irreversible dedication to tumorigenesis. Using single-cell transcriptomics, we find that Drosophila brain tumors contain a rapidly dividing stem cell population defined by upregulation of oxidative phosphorylation (OxPhos). We combine targeted metabolomics and in vivo genetic screening to demonstrate that OxPhos is required for tumor cell immortalization but dispensable in neural stem cells (NSCs) giving rise to tumors. Employing an in vivo NADH/NAD+ sensor, we show that NSCs precisely increase OxPhos during immortalization. Blocking OxPhos or mitochondrial fusion stalls TICs in quiescence and prevents tumorigenesis through impaired NAD+ regeneration. Our work establishes a unique connection between cellular metabolism and immortalization of tumor-initiating cells.
Subject(s)
Brain Neoplasms/metabolism , Carcinogenesis/metabolism , Cell Transformation, Neoplastic/metabolism , Mitochondrial Dynamics , NAD/metabolism , Neoplastic Stem Cells/metabolism , Neural Stem Cells/metabolism , Oxidative Phosphorylation , Animals , Brain Neoplasms/genetics , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Carcinogenesis/genetics , Carcinogenesis/pathology , Cell Transformation, Neoplastic/pathology , Citric Acid Cycle/genetics , Computational Biology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Glycolysis/genetics , Mass Spectrometry , Metabolomics , Microscopy, Electron, Transmission , Multigene Family , Neural Stem Cells/pathology , Oxygen Consumption/genetics , RNA Interference , Reactive Oxygen Species/metabolism , Single-Cell Analysis , Transcriptome/geneticsABSTRACT
Stem cells are highly abundant during early development but become a rare population in most adult organs. The molecular mechanisms causing stem cells to exit proliferation at a specific time are not well understood. Here, we show that changes in energy metabolism induced by the steroid hormone ecdysone and the Mediator initiate an irreversible cascade of events leading to cell-cycle exit in Drosophila neural stem cells. We show that the timely induction of oxidative phosphorylation and the mitochondrial respiratory chain are required in neuroblasts to uncouple the cell cycle from cell growth. This results in a progressive reduction in neuroblast cell size and ultimately in terminal differentiation. Brain tumor mutant neuroblasts fail to undergo this shrinkage process and continue to proliferate until adulthood. Our findings show that cell size control can be modified by systemic hormonal signaling and reveal a unique connection between metabolism and proliferation in stem cells.
Subject(s)
Cell Proliferation , Drosophila melanogaster/cytology , Ecdysone/metabolism , Neural Stem Cells/cytology , Animals , Cell Size , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Energy Metabolism , Genome, Insect , Mediator Complex/metabolism , Neural Stem Cells/metabolismABSTRACT
Stem cells need to balance self-renewal and differentiation for correct tissue development and homeostasis. Defects in this balance can lead to developmental defects or tumor formation. In recent years, mRNA splicing has emerged as an important mechanism regulating cell fate decisions. Here we address the role of the evolutionarily conserved splicing co-factor Barricade (Barc)/Tat-SF1/CUS2 in Drosophila neural stem cell (neuroblast) lineage formation. We show that Barc is required for the generation of neurons during Drosophila brain development by ensuring correct neural progenitor proliferation and differentiation. Barc associates with components of the U2 small nuclear ribonucleoprotein (snRNP) complex, and its depletion causes alternative splicing in the form of intron retention in a subset of genes. Using bioinformatics analysis and a cell culture-based splicing assay, we found that Barc-dependent introns share three major traits: they are short, GC rich and have weak 3' splice sites. Our results show that Barc, together with the U2 snRNP complex, plays an important role in regulating neural stem cell lineage progression during brain development and facilitates correct splicing of a subset of introns.
Subject(s)
Cell Cycle , Cell Lineage , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Transcription Factors/metabolism , Alternative Splicing/genetics , Animals , Base Composition/genetics , Base Sequence , Body Patterning/genetics , Brain/anatomy & histology , Cell Count , Cell Proliferation , Clone Cells , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Knockdown Techniques , Introns/genetics , Mice , Models, Biological , Mutation/genetics , Neurons/cytology , Neurons/metabolism , Phenotype , Protein Binding , RNA Interference , RNA Splice Sites/genetics , Ribonucleoprotein, U2 Small Nuclear/metabolism , Time FactorsABSTRACT
The TRIM-NHL protein Brain tumor (Brat) acts as a tumor suppressor in the Drosophila brain, but how it suppresses tumor formation is not completely understood. Here, we combine temperature-controlled brat RNAi with transcriptome analysis to identify the immediate Brat targets in Drosophila neuroblasts. Besides the known target Deadpan (Dpn), our experiments identify the transcription factor Zelda (Zld) as a critical target of Brat. Our data show that Zld is expressed in neuroblasts and required to allow re-expression of Dpn in transit-amplifying intermediate neural progenitors. Upon neuroblast division, Brat is enriched in one daughter cell where its NHL domain directly binds to specific motifs in the 3'UTR of dpn and zld mRNA to mediate their degradation. In brat mutants, both Dpn and Zld continue to be expressed, but inhibition of either transcription factor prevents tumorigenesis. Our genetic and biochemical data indicate that Dpn inhibition requires higher Brat levels than Zld inhibition and suggest a model where stepwise post-transcriptional inhibition of distinct factors ensures sequential generation of fates in a stem cell lineage.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinogenesis/genetics , Cell Lineage/genetics , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Neural Stem Cells/metabolism , Nuclear Proteins/genetics , Transcription Factors/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Basic Helix-Loop-Helix Transcription Factors/metabolism , Brain/metabolism , Brain/pathology , CRISPR-Cas Systems , Carcinogenesis/metabolism , Carcinogenesis/pathology , Cell Differentiation , DNA-Binding Proteins/metabolism , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Gene Editing , Gene Expression Regulation , Larva/genetics , Larva/growth & development , Larva/metabolism , Neural Stem Cells/pathology , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Proteolysis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolismABSTRACT
Tumor cells display features that are not found in healthy cells. How they become immortal and how their specific features can be exploited to combat tumorigenesis are key questions in tumor biology. Here we describe the long non-coding RNA cherub that is critically required for the development of brain tumors in Drosophila but is dispensable for normal development. In mitotic Drosophila neural stem cells, cherub localizes to the cell periphery and segregates into the differentiating daughter cell. During tumorigenesis, de-differentiation of cherub-high cells leads to the formation of tumorigenic stem cells that accumulate abnormally high cherub levels. We show that cherub establishes a molecular link between the RNA-binding proteins Staufen and Syncrip. As Syncrip is part of the molecular machinery specifying temporal identity in neural stem cells, we propose that tumor cells proliferate indefinitely, because cherub accumulation no longer allows them to complete their temporal neurogenesis program.
Subject(s)
Brain Neoplasms/pathology , Cell Transformation, Neoplastic , Neoplastic Stem Cells/physiology , Neural Stem Cells/physiology , RNA, Long Noncoding/metabolism , Animals , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Drosophila , Drosophila Proteins/metabolism , RNA, Long Noncoding/genetics , RNA-Binding Proteins/metabolismABSTRACT
Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi) or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a, and the previously uncharacterized gene MESR4, in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola) gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.