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.

dTcf/Pangolin suppresses growth and tumor formation in Drosophila.

Wnt/Wingless (Wg) signaling controls many aspects of animal development and is deregulated in different human cancers. The transcription factor dTcf/Pangolin (Pan) is the final effector of the Wg pathway in Drosophila and has a dual role in regulating the expression of Wg target genes. In the presence of Wg, dTcf/Pan interacts with ß-catenin/Armadillo (Arm) and induces the transcription of Wg targets. In absence of Wg, dTcf/Pan partners with the transcriptional corepressor TLE/Groucho (Gro) and inhibits gene expression. Here, we use the wing imaginal disk of Drosophila as a model to examine the functions that dTcf/Pan plays in a proliferating epithelium. We report a function of dTcf/Pan in growth control and tumorigenesis. Our results show that dTcf/Pan can limit tissue growth in normal development and suppresses tumorigenesis in the context of oncogene up-regulation. We identify the conserved transcription factors Sox box protein 15 (Sox15) and Ftz transcription factor 1 (Ftz-f1) as genes controlled by dTcf/Pan involved in tumor development. In conclusion, this study reports a role for dTcf/Pan as a repressor of normal and oncogenic growth and identifies the genes inducing tumorigenesis downstream of dTcf/Pan.

Pgc suppresses the zygotically acting RNA decay pathway to protect germ plasm RNAs in the Drosophila embryo.

Specification of germ cells is pivotal to ensure continuation of animal species. In many animal embryos, germ cell specification depends on maternally supplied determinants in the germ plasm. Drosophila polar granule component (pgc) mRNA is a component of the germ plasm. pgc encodes a small protein that is transiently expressed in newly formed pole cells, the germline progenitors, where it globally represses mRNA transcription. pgc is also required for pole cell survival, but the mechanism linking transcriptional repression to pole cell survival remains elusive. We report that pole cells lacking pgc show premature loss of germ plasm mRNAs, including the germ cell survival factor nanos, and undergo apoptosis. We found that pgc- pole cells misexpress multiple miRNA genes. Reduction of miRNA pathway activity in pgc- embryos partially suppressed germ plasm mRNA degradation and pole cell death, suggesting that Pgc represses zygotic miRNA transcription in pole cells to protect germ plasm mRNAs. Interestingly, germ plasm mRNAs are protected from miRNA-mediated degradation in vertebrates, albeit by a different mechanism. Thus, independently evolved mechanisms are used to silence miRNAs during germ cell specification.

Identification and characterization of novel conserved RNA structures in Drosophila.

BACKGROUND: Comparative genomics approaches have facilitated the discovery of many novel non-coding and structured RNAs (ncRNAs). The increasing availability of related genomes now makes it possible to systematically search for compensatory base changes - and thus for conserved secondary structures - even in genomic regions that are poorly alignable in the primary sequence. The wealth of available transcriptome data can add valuable insight into expression and possible function for new ncRNA candidates. Earlier work identifying ncRNAs in Drosophila melanogaster made use of sequence-based alignments and employed a sliding window approach, inevitably biasing identification toward RNAs encoded in the more conserved parts of the genome. RESULTS: To search for conserved RNA structures (CRSs) that may not be highly conserved in sequence and to assess the expression of CRSs, we conducted a genome-wide structural alignment screen of 27 insect genomes including D. melanogaster and integrated this with an extensive set of tiling array data. The structural alignment screen revealed ∼30,000 novel candidate CRSs at an estimated false discovery rate of less than 10%. With more than one quarter of all individual CRS motifs showing sequence identities below 60%, the predicted CRSs largely complement the findings of sliding window approaches applied previously. While a sixth of the CRSs were ubiquitously expressed, we found that most were expressed in specific developmental stages or cell lines. Notably, most statistically significant enrichment of CRSs were observed in pupae, mainly in exons of untranslated regions, promotors, enhancers, and long ncRNAs. Interestingly, cell lines were found to express a different set of CRSs than were found in vivo. Only a small fraction of intergenic CRSs were co-expressed with the adjacent protein coding genes, which suggests that most intergenic CRSs are independent genetic units. CONCLUSIONS: This study provides a more comprehensive view of the ncRNA transcriptome in fly as well as evidence for differential expression of CRSs during development and in cell lines.

Metabolic control of PPAR activity by aldehyde dehydrogenase regulates invasive cell behavior and predicts survival in hepatocellular and renal clear cell carcinoma.

BACKGROUND: Changes in cellular metabolism are now recognized as potential drivers of cancer development, rather than as secondary consequences of disease. Here, we explore the mechanism by which metabolic changes dependent on aldehyde dehydrogenase impact cancer development. METHODS: ALDH7A1 was identified as a potential cancer gene using a Drosophila in vivo metastasis model. The role of the human ortholog was examined using RNA interference in cell-based assays of cell migration and invasion. 1H-NMR metabolite profiling was used to identify metabolic changes in ALDH7A1-depleted cells. Publically available cancer gene expression data was interrogated to identify a gene-expression signature associated with depletion of ALDH7A1. Computational pathway and gene set enrichment analysis was used to identify signaling pathways and cellular processes that were correlated with reduced ALDH7A1 expression in cancer. A variety of statistical tests used to evaluate these analyses are described in detail in the methods section. Immunohistochemistry was used to assess ALDH7A1 expression in tissue samples from cancer patients. RESULTS: Depletion of ALDH7A1 increased cellular migration and invasiveness in vitro. Depletion of ALDH7A1 led to reduced levels of metabolites identified as ligands for Peroxisome proliferator-activated receptor (PPARα). Analysis of publically available cancer gene expression data revealed that ALDH7A1 mRNA levels were reduced in many human cancers, and that this correlated with poor survival in kidney and liver cancer patients. Using pathway and gene set enrichment analysis, we establish a correlation between low ALDH7A1 levels, reduced PPAR signaling and reduced patient survival. Metabolic profiling showed that endogenous PPARα ligands were reduced in ALDH7A1-depleted cells. ALDH7A1-depletion led to reduced PPAR transcriptional activity. Treatment with a PPARα agonist restored normal cellular behavior. Low ALDH7A1 protein levels correlated with poor clinical outcome in hepatocellular and renal clear cell carcinoma patients. CONCLUSIONS: We provide evidence that low ALDH7A1 expression is a useful prognostic marker of poor clinical outcome for hepatocellular and renal clear cell carcinomas and hypothesize that patients with low ALDH7A1 might benefit from therapeutic approaches addressing PPARα activity.

Warburg Effect Metabolism Drives Neoplasia in a Drosophila Genetic Model of Epithelial Cancer.

Cancers develop in a complex mutational landscape. Genetic models of tumor formation have been used to explore how combinations of mutations cooperate to promote tumor formation in vivo. Here, we identify lactate dehydrogenase (LDH), a key enzyme in Warburg effect metabolism, as a cooperating factor that is both necessary and sufficient for epidermal growth factor receptor (EGFR)-driven epithelial neoplasia and metastasis in a Drosophila model. LDH is upregulated during the transition from hyperplasia to neoplasia, and neoplasia is prevented by LDH depletion. Elevated LDH is sufficient to drive this transition. Notably, genetic alterations that increase glucose flux, or a high-sugar diet, are also sufficient to promote EGFR-driven neoplasia, and this depends on LDH activity. We provide evidence that increased LDHA expression promotes a transformed phenotype in a human primary breast cell culture model. Furthermore, analysis of publically available cancer data showed evidence of synergy between elevated EGFR and LDHA activity linked to poor clinical outcome in a number of human cancers. Altered metabolism has generally been assumed to be an enabling feature that accelerates cancer cell proliferation. Our findings provide evidence that sugar metabolism may have a more profound role in driving neoplasia than previously appreciated.

USP21 regulates Hippo pathway activity by mediating MARK protein turnover.

The Hippo pathway, which acts to repress the activity of YAP and TAZ trancriptional co-activators, serve as a barrier for oncogenic transformation. Unlike other oncoproteins, YAP and TAZ are rarely activated by mutations or amplified in cancer. However, elevated YAP/TAZ activity is frequently observed in cancer and often correlates with worse survival. The activity and stability of Hippo pathway components, including YAP/TAZ, AMOT and LATS1/2, are regulated by ubiquitin-mediated protein degradation. Aberrant expression of ubiquitin ligase complexes that regulate the turnover of Hippo components and deubiquitylating enzymes that counteract these ubiquitin ligases have been implicated in human cancer. Here we identify the USP21 deubiquitylating enzyme as a novel regulator of Hippo pathway activity. We provide evidence that USP21 regulates YAP/TAZ activity by controlling the stability of MARK kinases, which promote Hippo signaling. Low expression of USP21 in early stage renal clear cell carcinoma suggests that USP21 may be a useful biomarker.

The chromatin remodeling BAP complex limits tumor-promoting activity of the Hippo pathway effector Yki to prevent neoplastic transformation in Drosophila epithelia.

Switch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes are mutated in many human cancers. In this article, we make use of a Drosophila genetic model for epithelial tumor formation to explore the tumor suppressive role of SWI/SNF complex proteins. Members of the BAP complex exhibit tumor suppressor activity in tissue overexpressing the Yorkie (Yki) proto-oncogene, but not in tissue overexpressing epidermal growth factor receptor (EGFR). The Brahma-associated protein (BAP) complex has been reported to serve as a Yki-binding cofactor to support Yki target expression. However, we observed that depletion of BAP leads to ectopic expression of Yki targets both autonomously and non-autonomously, suggesting additional indirect effects. We provide evidence that BAP complex depletion causes upregulation of the Wingless (Wg) and Decapentaplegic (Dpp) morphogens to promote tumor formation in cooperation with Yki.

DUB3 Deubiquitylating Enzymes Regulate Hippo Pathway Activity by Regulating the Stability of ITCH, LATS and AMOT Proteins.

The YAP and TAZ transcriptional coactivators promote oncogenic transformation. Elevated YAP/TAZ activity has been documented in human tumors. YAP and TAZ are negatively regulated by the Hippo tumor suppressor pathway. The activity and stability of several Hippo pathway components, including YAP/TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor by limiting YAP activity.

Drosophila as a Model to Study the Link between Metabolism and Cancer.

Cellular metabolism has recently been recognized as a hallmark of cancer. Investigating the origin and effects of the reprogrammed metabolism of tumor cells, and identifying its genetic mediators, will improve our understanding of how these changes contribute to disease progression and may suggest new approaches to therapy. Drosophila melanogaster is emerging as a valuable model to study multiple aspects of tumor formation and malignant transformation. In this review, we discuss the use of Drosophila as model to study how changes in cellular metabolism, as well as metabolic disease, contribute to cancer.

Deubiquitylating enzyme USP9x regulates hippo pathway activity by controlling angiomotin protein turnover.

The Hippo pathway has been identified as a key barrier for tumorigenesis, acting through downregulation of YAP/TAZ activity. Elevated YAP/TAZ activity has been documented in many human cancers. Ubiquitylation has been shown to play a key role in regulating YAP/TAZ activity through downregulation of a number of Hippo pathway components. Several ubiquitin ligase complexes have been implicated in this process, however, little is known about the deubiquitylating enzymes that counteract these activities to regulate YAP/TAZ. Here we identify the deubiquitylating enzyme USP9x as a regulator of YAP/TAZ activity. We demonstrate that USPx regulates ubiquitin-mediated turnover of the YAP inhibitor, Angiomotin. USP9x acts to deubiquitylate Angiomotin at lysine 496, resulting in stabilization of Angiomotin and lower YAP/TAZ activity. USP9x mRNA levels were reduced in several cancers. Clinically, USP9x mRNA levels were reduced in several cancers with low USPx expression correlating with poor prognosis in renal clear cell carcinoma. Our data indicate that USP9x may be a useful biomarker for renal clear cell carcinoma.

Cancer in Drosophila: Imaginal Discs as a Model for Epithelial Tumor Formation.

Cancer genomics has greatly increased our understanding of the complexity of the genetic and epigenetic changes found in human tumors. Understanding the functional relationships among these elements calls for the use of flexible genetic models. We discuss the use of Drosophila models to study cooperation among genetic factors that contribute to disease progression.

A conformation-induced fluorescence method for microRNA detection.

MicroRNAs play important roles in a large variety of biological systems and processes through their regulation of target mRNA expression, and show promise as clinical biomarkers. However, their small size presents challenges for tagging or direct detection. Innovation in techniques to sense and quantify microRNAs may aid research into novel aspects of microRNA biology and contribute to the development of diagnostics. By introducing an additional stem loop into the fluorescent RNA Spinach and altering its 3' and 5' ends, we have generated a new RNA, Pandan, that functions as the basis for a microRNA sensor. Pandan contains two sequence-variable stem loops that encode complementary sequence for a target microRNA of interest. In its sensor form, it requires the binding of a target microRNA in order to reconstitute the RNA scaffold for fluorophore binding and fluorescence. Binding of the target microRNA resulted in large changes in fluorescence intensity. The median fold change in fluorescence observed for the sensors tested was ∼50-fold. Pandan RNA sensors exhibit good signal-to-noise ratios, and can detect their target microRNAs within complex RNA mixtures.

Cell Competition Drives the Formation of Metastatic Tumors in a Drosophila Model of Epithelial Tumor Formation.

Cell competition is a homeostatic process in which proliferating cells compete for survival. Elimination of otherwise normal healthy cells through competition is important during development and has recently been shown to contribute to maintaining tissue health during organismal aging. The mechanisms that allow for ongoing cell competition during adult life could, in principle, contribute to tumorigenesis. However, direct evidence supporting this hypothesis has been lacking. Here, we provide evidence that cell competition drives tumor formation in a Drosophila model of epithelial cancer. Cells expressing EGFR together with the conserved microRNA miR-8 acquire the properties of supercompetitors. Neoplastic transformation and metastasis depend on the ability of these cells to induce apoptosis and engulf nearby cells. miR-8 expression causes genome instability by downregulating expression of the Septin family protein Peanut. Cytokinesis failure due to downregulation of Peanut is required for tumorigenesis. This study provides evidence that the cellular mechanisms that drive cell competition during normal tissue growth can be co-opted to drive tumor formation and metastasis. Analogous mechanisms for cytokinesis failure may lead to polyploid intermediates in tumorigenesis in mammalian cancer models.

Control of Drosophila Type I and Type II central brain neuroblast proliferation by bantam microRNA.

Post-transcriptional regulation of stem cell self-renewal by microRNAs is emerging as an important mechanism controlling tissue homeostasis. Here, we provide evidence that bantam microRNA controls neuroblast number and proliferation in the Drosophila central brain. Bantam also supports proliferation of transit-amplifying intermediate neural progenitor cells in type II neuroblast lineages. The stem cell factors brat and prospero are identified as bantam targets acting on different aspects of these processes. Thus, bantam appears to act in multiple regulatory steps in the maintenance and proliferation of neuroblasts and their progeny to regulate growth of the central brain.

miR-965 controls cell proliferation and migration during tissue morphogenesis in the Drosophila abdomen.

Formation of the Drosophila adult abdomen involves a process of tissue replacement in which larval epidermal cells are replaced by adult cells. The progenitors of the adult epidermis are specified during embryogenesis and, unlike the imaginal discs that make up the thoracic and head segments, they remain quiescent during larval development. During pupal development, the abdominal histoblast cells proliferate and migrate to replace the larval epidermis. Here, we provide evidence that the microRNA, miR-965, acts via string and wingless to control histoblast proliferation and migration. Ecdysone signaling downregulates miR-965 at the onset of pupariation, linking activation of the histoblast nests to the hormonal control of metamorphosis. Replacement of the larval epidermis by adult epidermal progenitors involves regulation of both cell-intrinsic events and cell communication. By regulating both cell proliferation and cell migration, miR-965 contributes to the robustness of this morphogenetic system.

Regulation of pattern formation and gene amplification during Drosophila oogenesis by the miR-318 microRNA.

Pattern formation during epithelial development requires the coordination of multiple signaling pathways. Here, we investigate the functions of an ovary-enriched miRNA, miR-318, in epithelial development during Drosophila oogenesis. mir-318 maternal loss-of-function mutants were female-sterile and laid eggs with abnormal morphology. Removal of mir-318 disrupted the dorsal-anterior follicle cell patterning, resulting in abnormal dorsal appendages. mir-318 mutant females also produced thin and fragile eggshells due to impaired chorion gene amplification. We provide evidence that the ecdysone signaling pathway activates expression of miR-318 and that miR-318 cooperates with Tramtrack69 to control the switch from endocycling to chorion gene amplification during differentiation of the follicular epithelium. The multiple functions of miR-318 in oogenesis illustrate the importance of miRNAs in maintaining cell fate and in promoting the developmental transition in the female follicular epithelium.

A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate excitotoxicity.

Evidence has begun to emerge for microRNAs as regulators of synaptic signaling, specifically acting to control postsynaptic responsiveness during synaptic transmission. In this report, we provide evidence that Drosophila melanogaster miR-1000 acts presynaptically to regulate glutamate release at the synapse by controlling expression of the vesicular glutamate transporter (VGlut). Genetic deletion of miR-1000 led to elevated apoptosis in the brain as a result of glutamatergic excitotoxicity. The seed-similar miR-137 regulated VGluT2 expression in mouse neurons. These conserved miRNAs share a neuroprotective function in the brains of flies and mice. Drosophila miR-1000 showed activity-dependent expression, which might serve as a mechanism to allow neuronal activity to fine-tune the strength of excitatory synaptic transmission.

Feedback regulation on PTEN/AKT pathway by the ER stress kinase PERK mediated by interaction with the Vault complex.

The high proliferation rate of cancer cells, together with environmental factors such as hypoxia and nutrient deprivation can cause Endoplasmic Reticulum (ER) stress. The protein kinase PERK is an essential mediator in one of the three ER stress response pathways. Genetic and pharmacological inhibition of PERK has been reported to limit tumor growth in xenograft models. Here we provide evidence that inactive PERK interacts with the nuclear pore-associated Vault complex protein and that this compromises Vault-mediated nuclear transport of PTEN. Pharmacological inhibition of PERK under ER stress results is abnormal sequestration of the Vault complex, leading to increased cytoplasmic PTEN activity and lower AKT activation. As the PI3K/PTEN/AKT pathway is crucial for many aspects of cell growth and survival, this unexpected effect of PERK inhibitors on AKT activity may have implications for their potential use as therapeutic agents.

Systematic study of Drosophila microRNA functions using a collection of targeted knockout mutations.

MicroRNAs are abundant in animal genomes, yet little is known about their functions in vivo. Here, we report the production of 80 new Drosophila miRNA mutants by targeted homologous recombination. These mutants remove 104 miRNAs. Together with 15 previously reported mutants, this collection includes 95 mutants deleting 130 miRNAs. Collectively, these genes produce over 99% of all Drosophila miRNAs, measured by miRNA sequence reads. We present a survey of developmental and adult miRNA phenotypes. Over 80% of the mutants showed at least one phenotype using a p < 0.01 significance threshold. We observed a significant correlation between miRNA abundance and phenotypes related to survival and lifespan, but not to most other phenotypes. miRNA cluster mutants were no more likely than single miRNA mutants to produce significant phenotypes. This mutant collection will provide a resource for future analysis of the biological roles of Drosophila miRNAs.