Genome-Wide Screen for Context-Dependent Tumor Suppressors Identified Using in Vivo Models for Neoplasia in Drosophila.

Genetic approaches in Drosophila have successfully identified many genes involved in regulation of growth control as well as genetic interactions relevant to the initiation and progression of cancer in vivo Here, we report on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: the Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo We also report on an independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. We also find a number of novel connections to Yki and EGFR driven tissue growth, mostly unique to one of the two. Thus, resources provided here would be useful to all researchers who study negative regulators of growth during development and cancer in the context of activated EGFR and/or Yki and positive regulators of growth in the context of activated EGFR. Resources reported here are available freely for anyone to use.

Drosophila neuroblasts as a new model for the study of stem cell self-renewal and tumour formation.

Drosophila larval brain stem cells (neuroblasts) have emerged as an important model for the study of stem cell asymmetric division and the mechanisms underlying the transformation of neural stem cells into tumour-forming cancer stem cells. Each Drosophila neuroblast divides asymmetrically to produce a larger daughter cell that retains neuroblast identity, and a smaller daughter cell that is committed to undergo differentiation. Neuroblast self-renewal and differentiation are tightly controlled by a set of intrinsic factors that regulate ACD (asymmetric cell division). Any disruption of these two processes may deleteriously affect the delicate balance between neuroblast self-renewal and progenitor cell fate specification and differentiation, causing neuroblast overgrowth and ultimately lead to tumour formation in the fly. In this review, we discuss the mechanisms underlying Drosophila neural stem cell self-renewal and differentiation. Furthermore, we highlight emerging evidence in support of the notion that defects in ACD in mammalian systems, which may play significant roles in the series of pathogenic events leading to the development of brain cancers.

Protein trafficking abnormalities in Drosophila tissues with impaired activity of the ZIP7 zinc transporter Catsup.

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of the Notch signaling pathway is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is crucial for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila epithelial tissues, we recovered mutations that disrupt the Catsup gene, which encodes the Drosophila ortholog of the mammalian ZIP7 zinc transporter. Loss of Catsup function causes Notch to accumulate abnormally in the endoplasmic reticulum (ER) and Golgi compartments, resulting in impaired Notch signaling. In addition, Catsup mutant cells exhibit elevated ER stress, suggesting that impaired zinc homeostasis causes increased levels of misfolded proteins within the secretory compartment.

Therapeutic approaches to modulating Notch signaling: current challenges and future prospects.

Dysregulated Notch signaling has been implicated in numerous human diseases, including a broad spectrum of cancers. Mutations in Notch1 are prevalent in T-cell acute lymphoblastic leukemia, and abnormal expression of different human Notch receptors contributes to B-cell tumors as well as cancers of the breast, lung, pancreas, skin, prostate, colon, brain and other tissues. Several γ-secretase inhibitors, small chemical compounds that were initially developed to inhibit the activity of the γ-secretase aspartyl protease in Alzheimer's disease, are now being explored for their potential chemotherapeutic applications in Notch-associated cancers. An alternative approach involves the development of antibodies to inhibit specific Notch receptors, their activating ligands, or other components of the Notch pathway in tumors. Here we review recent progress and current challenges in the use of these strategies to modulate Notch signaling for cancer therapy.

Pharmacological analysis of Drosophila melanogaster gamma-secretase with respect to differential proteolysis of Notch and APP.

The gamma-secretase aspartyl protease is responsible for the cleavage of numerous type I integral membrane proteins, including amyloid precursor protein (APP) and Notch. APP cleavage contributes to the generation of toxic amyloid beta peptides in Alzheimer's disease, whereas cleavage of the Notch receptor is required for normal physiological signaling between differentiating cells. Mutagenesis studies as well as in vivo analyses of Notch and APP activity in the presence of pharmacological inhibitors indicate that these substrates can be differentially modulated by inhibition of mammalian gamma-secretase, although some biochemical studies instead show nearly identical dose-response inhibitor effects on Notch and APP cleavages. Here, we examine the dose-response effects of several inhibitors on Notch and APP in Drosophila melanogaster cells, which possess a homogeneous form of gamma-secretase. Four different inhibitors that target different domains of gamma-secretase exhibit similar dose-response effects for both substrates, including rank order of inhibitor potencies and effective concentration ranges. For two inhibitors, modest differences in inhibitor dose responses toward Notch and APP were detected, suggesting that inhibitors might be identified that possess some discrimination in their ability to target alternative gamma-secretase substrates. These findings also indicate that despite an overall conservation in inhibitor potencies toward different gamma-secretase substrates, quantitative differences might exist that could be relevant for the development of therapeutically valuable substrate-specific inhibitors.

Zebrafish fgfr1 is a member of the fgf8 synexpression group and is required for fgf8 signalling at the midbrain-hindbrain boundary.

FGFR1 is an important signalling molecule during embryogenesis and in adulthood. FGFR1 mutations in human may lead to developmental defects and pathological conditions, including cancer and Alzheimer's disease. Here, we describe cloning and expression analysis of the zebrafish fibroblast growth factor receptor 1 ( fgfr1). Initially, fgfr1 is expressed in the adaxial mesoderm with transcripts distinctly localised to the anterior portion of each half-somite. Hereupon, fgfr1 is also strongly expressed in the otic vesicles, branchial arches and the brain, especially at the midbrain-hindbrain boundary (MHB). The expression patterns of fgfr1 and fgf8 are strikingly similar and knock-down of fgfr1 phenocopies many aspects observed in the fgf8 mutant acerebellar, suggesting that Fgf8 exerts its function mainly by binding to FgfR1.

Developmental control of Presenilin1 expression, endoproteolysis, and interaction in zebrafish embryos.

Dominant mutations in presenilin1 (PS1) and presenilin2 (PS2) are a major cause of early-onset Alzheimer's disease. In this report we analyze the expression of the zebrafish presenilin1 (Psen1) and presenilin2 (Psen2) proteins during embryogenesis. We demonstrate that Psen1 and Psen2 holoproteins are relatively abundant in zebrafish embryos and are proteolytically processed. Psen1 is maternally expressed, whereas Psen2 is expressed at later stages during development. The Psen1 C-terminal proteolytic fragment (CTF) is present at varying levels during embryogenesis, indicating the existence of developmental control mechanisms regulating its production. We examine the codependency of Psen1 and Psen2 expression during early embryogenesis. Forced overexpression of psen2 increases expression of Psen2 holoprotein, but not the N-terminal fragment (NTF), indicating that levels of Psen2 NTF are strictly controlled. Overexpression of psen2 did not alter levels of Psen1 holoprotein, CTF, or higher molecular weight complexes. Reduction of Psen1 activity in zebrafish embryos produces similar developmental defects to those seen for loss of PS1 activity in knockout mice. The relevance of these results to previous work on presenilin protein regulation and function are discussed. Our work shows that zebrafish embryos are a valid and valuable system in which to study presenilin interactions, regulation, and function.

High-rate evolution of Saccharomyces sensu lato chromosomes.

Forty isolates belonging to the Saccharomyces sensu lato complex were analyzed for one nuclear and two mitochondrial sequences, and for their karyotypes. These data are useful for description and definition of yeast species based on the phylogenetic species concept. The deduced phylogenetic relationships among isolates based on the nuclear and mitochondrial sequences were usually similar, suggesting that horizontal transfer/introgression has not been frequent. The highest degree of polymorphism was observed at the chromosome level. Even isolates which had identical nuclear and mitochondrial sequences often exhibited variation in the number and size of their chromosomes. Apparently, yeast chromosomes have been frequently reshaped and therefore also the position of genes has been dynamic during the evolutionary history of yeasts.

Identification of a second presenilin gene in zebrafish with similarity to the human Alzheimer's disease gene presenilin2.

Presenilins play prominent roles in the molecular pathogenesis of Alzheimer's disease and during embryo development. We have isolated a zebrafish presenilin orthologue (pre2), which shows a high degree of sequence identity to the human PS2 protein. Zebrafish pre2 is maternally and ubiquitously expressed during early embryo development, whereas Pre2 protein expression is initiated between 6 and 12 hours post fertilisation (hpf), suggesting strict regulation of pre2 translation. pre2 expression is especially high in neural-crest-derived melanocytes.

The structure and function of vertebrate fibroblast growth factor receptor 1.

The vertebrate fibroblast growth factor receptor 1 (FGFR1) is alternatively spliced generating multiple splice variants that are differentially expressed during embryo development and in the adult body. The restricted expression patterns of FGFR1 isoforms, together with differential expression and binding of specific ligands, leads to activation of common FGFR1 signal transduction pathways, but may result in distinctively different biological responses as a result of differences in cellular context. FGFR1 isoforms are also present in the nucleus in complex with various fibroblast growth factors where they function to regulate transcription of target genes.