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MiR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila.

ABSTRACT: Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.

SUBMITTER: Daniel SG

PROVIDER: S-EPMC5829493 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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miR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila.

Daniel Scott G SG Russ Atlantis D AD Guthridge Kathryn M KM Raina Ammad I AI Estes Patricia S PS Parsons Linda M LM Richardson Helena E HE Schroeder Joyce A JA Zarnescu Daniela C DC

Biology open 20180126 1

Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role ...[more]

PMID: 29361610

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Project description:Cancer is a complex phenomenon involving multiple cellular pathways and processes in various combinations. As a genetic system, Drosophila has contributed much to our understanding of processes that initiate tumorigenesis. However, these are still disparate threads of information and a comprehensive understanding of how these various events and processes come together to bring about tumorous transformation is still wanting. The present study is an attempt to create a comprehensive picture of the dynamic process of epithelial tumour development in imaginal discs using lgl mutants as a model of neoplastic transformation. Experimental Procedure: The experiments were carried out in isogenized w-; lgl4. FRT40A/CyO. Actin –GFP (E Gateff and Schneiderman 1974). Fairly homogenous cultures of flies were added to population cages 2-3 days post eclosion for egg collection. Eggs were collected on yeasted agar plates which were kept in the cages for 1 to 1.5 hours. Plates with eggs were kept at 25 degrees for 21 hours, after which they were gently washed under running water to remove early hatched larvae. The plates were then yeasted again and kept at 25 degrees for another 24 hours to let all the eggs hatch. The food with the 24 hour old larvae was then transferred to large food-grade plastic boxes that had a single cotton plugged hole on the lid to allow for proper ventilation. These boxes also had standard fly food and were well yeasted. The larvae were allowed to grow in these boxes at 25 degrees until the time they were harvested. No other synchronization process was done on these cultures. The boxes containing synchronized larval cultures were washed in gently running water and the water along with the washed off larvae were collected in a beaker. The larvae were allowed to sink to the bottom and the water was decanted off. This was repeated many times till the decanted water was clear of food and other debris. Finally, a 3M solution of NaCl was poured into the beaker. This causes the larvae to float to the surface along with the large pieces of food left over from the water washes. Water was then gently added to the beaker till the concentration of the salt solution was just enough to allow only the larvae to float on the surface while the food pieces sunk to the bottom. At this point, the solution, along with the floating larvae was decanted off through a sieve to collect the larvae. These were then transferred to a glass petri-plate and washed with distilled water to remove all traces of salt solution. The larvae were finally floated in PBS for GFP selection under fluorescence microscope. The selected larvae were transferred to glass cavity blocks rinsed in RNAzap RNAse Inhibitor solution containing RNA-ase free PBS and dissected. For isolating imaginal discs, larvae were cut from the middle with the help of forceps and scissors, and then flipped inside out with needles so that the inner body wall is completely exposed with the imaginal discs attached. Fat bodies and other internal organs were carefully removed with the help of the forceps and needles. The imaginal discs/tumors were dissected out from these flipped larvae, care being taken to remove all traces of fat bodies or other debris from the discs and immediately transferred to RNAse free Microfuge tubes. The tubes were centrifuged briefly to collect the imaginal discs/tumors at the bottom while the PBS was pipetted off. The discs were then resuspended in 150-300 uL of RLT Buffer (Qiagen) in which 1.5-3 uL of Beta-mercaptoethanol was added. The tubes were then vortexed to lyse the cells. This suspension was stored at -40 degrees until RNA preparation. Total RNA was prepared from the lysed wing discs/tumors suspended in the guanidine thiocynate containing Lysis Buffer (RLT) supplied with the RNeasy Mini Kit and 14.3M beta-Mercaptoethanol using the ethanol precipitation/column filtration protocol recommended for the RNeasy Mini Kit by the Manufacturer. The purified total RNA was quantified and its quality checked by spectrophotometry and Agarose Gel electrophoresis respectively. Total RNA from 30-50 pairs of imaginal discs/tumors were used to make a single sample depending on the tumor stage with RNA yields typically ranging from 10 ug to 15 ug. Purified total RNA passing the quality controls were immediately used for labeling and hybridization reactions. The total RNA was processed and hybridized to Affymetrix GeneChip Drosophila Whole Genome 2.0 arrays using the manufacturer’s protocol, and the arrays were scanned immediately after hybridization.

Project description:BackgroundNeoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. Precancerous cells are often removed by cell death from normal tissues in the early steps of the tumourigenic process, but the molecules responsible for such a fundamental safeguard process remain in part elusive. With the aim to investigate the molecular crosstalk occurring between precancerous and normal cells in vivo, we took advantage of the clonal analysis methods that are available in Drosophila for studying the phenotypes due to lethal giant larvae (lgl) neoplastic mutation induced in different backgrounds and tissues.ResultsWe observed that lgl mutant cells growing in wild-type imaginal wing discs show poor viability and are eliminated by Jun N-terminal Kinase (JNK)-dependent cell death. Furthermore, they express very low levels of dMyc oncoprotein compared with those found in the surrounding normal tissue. Evidence that this is a cause of lgl mutant cells elimination was obtained by increasing dMyc levels in lgl mutant clones: their overgrowth potential was indeed re-established, with mutant cells overwhelming the neighbouring tissue and forming tumourous masses displaying several cancer hallmarks. Moreover, when lgl mutant clones were induced in backgrounds of slow-dividing cells, they upregulated dMyc, lost apical-basal cell polarity and were able to overgrow. Those phenotypes were abolished by reducing dMyc levels in the mutant clones, thereby confirming its key role in lgl-induced tumourigenesis. Furthermore, we show that the eiger-dependent Intrinsic Tumour Suppressor pathway plays only a minor role in eliminating lgl mutant cells in the wing pouch; lgl-/- clonal death in this region is instead driven mainly by dMyc-induced Cell Competition.ConclusionsOur results provide the first evidence that dMyc oncoprotein is required in lgl tumour suppressor mutant tissue to promote invasive overgrowth in larval and adult epithelial tissues. Moreover, we show that dMyc abundance inside versus outside the mutant clones plays a key role in driving neoplastic overgrowth.

Dataset Information

MiR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila.

Publications

<i>miR-9a</i> mediates the role of Lethal giant larvae as an epithelial growth inhibitor in <i>Drosophila</i>.

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