Myc-regulated miRNAs modulate p53 expression and impact animal survival under nutrient deprivation.

The conserved transcription factor Myc regulates cell growth, proliferation and apoptosis, and its deregulation has been associated with human pathologies. Although specific miRNAs have been identified as fundamental components of the Myc tumorigenic program, how Myc regulates miRNA biogenesis remains controversial. Here we showed that Myc functions as an important regulator of miRNA biogenesis in Drosophila by influencing both miRNA gene expression and processing. Through the analysis of ChIP-Seq datasets, we discovered that nearly 56% of Drosophila miRNA genes show dMyc binding, exhibiting either the canonical or non-canonical E-box sequences within the peak region. Consistently, reduction of dMyc levels resulted in widespread downregulation of miRNAs gene expression. dMyc also modulates miRNA processing and activity by controlling Drosha and AGO1 levels through direct transcriptional regulation. By using in vivo miRNA activity sensors we demonstrated that dMyc promotes miRNA-mediated silencing in different tissues, including the wing primordium and the fat body. We also showed that dMyc-dependent expression of miR-305 in the fat body modulates Dmp53 levels depending on nutrient availability, having a profound impact on the ability of the organism to respond to nutrient stress. Indeed, dMyc depletion in the fat body resulted in extended survival to nutrient deprivation which was reverted by expression of either miR-305 or a dominant negative version of Dmp53. Our study reveals a previously unrecognized function of dMyc as an important regulator of miRNA biogenesis and suggests that Myc-dependent expression of specific miRNAs may have important tissue-specific functions.

FOXO-mediated repression of Dicer1 regulates metabolism, stress resistance, and longevity in Drosophila.

The adipose tissue plays a crucial role in metabolism and physiology, affecting animal lifespan and susceptibility to disease. In this study, we present evidence that adipose Dicer1 (Dcr-1), a conserved type III endoribonuclease involved in miRNA processing, plays a crucial role in the regulation of metabolism, stress resistance, and longevity. Our results indicate that the expression of Dcr-1 in murine 3T3L1 adipocytes is responsive to changes in nutrient levels and is subject to tight regulation in the Drosophila fat body, analogous to human adipose and hepatic tissues, under various stress and physiological conditions such as starvation, oxidative stress, and aging. The specific depletion of Dcr-1 in the Drosophila fat body leads to changes in lipid metabolism, enhanced resistance to oxidative and nutritional stress, and is associated with a significant increase in lifespan. Moreover, we provide mechanistic evidence showing that the JNK-activated transcription factor FOXO binds to conserved DNA-binding sites in the dcr-1 promoter, directly repressing its expression in response to nutrient deprivation. Our findings emphasize the importance of FOXO in controlling nutrient responses in the fat body by suppressing Dcr-1 expression. This mechanism coupling nutrient status with miRNA biogenesis represents a novel and previously unappreciated function of the JNK-FOXO axis in physiological responses at the organismal level.

Fat Body p53 Regulates Systemic Insulin Signaling and Autophagy under Nutrient Stress via Drosophila Upd2 Repression.

The tumor suppressor p53 regulates multiple metabolic pathways at the cellular level. However, its role in the context of a whole animal response to metabolic stress is poorly understood. Using Drosophila, we show that AMP-activated protein kinase (AMPK)-dependent Dmp53 activation is critical for sensing nutrient stress, maintaining metabolic homeostasis, and extending organismal survival. Under both nutrient deprivation and high-sugar diet, Dmp53 activation in the fat body represses expression of the Drosophila Leptin analog, Unpaired-2 (Upd2), which remotely controls Dilp2 secretion in insulin-producing cells. In starved Dmp53-depleted animals, elevated Upd2 expression in adipose cells and activation of Upd2 receptor Domeless in the brain result in sustained Dilp2 circulating levels and impaired autophagy induction at a systemic level, thereby reducing nutrient stress survival. These findings demonstrate an essential role for the AMPK-Dmp53 axis in nutrient stress responses and expand the concept that adipose tissue acts as a sensing organ that orchestrates systemic adaptation to nutrient status.

Eiger/TNFα-mediated Dilp8 and ROS production coordinate intra-organ growth in Drosophila.

Coordinated intra- and inter-organ growth during animal development is essential to ensure a correctly proportioned individual. The Drosophila wing has been a valuable model system to reveal the existence of a stress response mechanism involved in the coordination of growth between adjacent cell populations and to identify a role of the fly orthologue of p53 (Dmp53) in this process. Here we identify the molecular mechanisms used by Dmp53 to regulate growth and proliferation in a non-autonomous manner. First, Dmp53-mediated transcriptional induction of Eiger, the fly orthologue of TNFα ligand, leads to the cell-autonomous activation of JNK. Second, two distinct signaling events downstream of the Eiger/JNK axis are induced in order to independently regulate tissue size and cell number in adjacent cell populations. Whereas expression of the hormone dILP8 acts systemically to reduce growth rates and tissue size of adjacent cell populations, the production of Reactive Oxygen Species-downstream of Eiger/JNK and as a consequence of apoptosis induction-acts in a non-cell-autonomous manner to reduce proliferation rates. Our results unravel how local and systemic signals act concertedly within a tissue to coordinate growth and proliferation, thereby generating well-proportioned organs and functionally integrated adults.

Regulation and function of p53: A perspective from Drosophila studies.

Tp53 is a central regulator of cellular responses to stress and one of the most frequently mutated genes in human cancers. P53 is activated by a myriad of stress signals and drives specific cellular responses depending on stress nature, cell type and cellular context. Additionally to its classical functions in regulating cell cycle arrest, apoptosis and senescence, newly described non-canonical functions of p53 are increasingly coming under the spotlight as important functions not only for its role as a tumour suppressor but also for its non-cancer associated activities. Drosophila melanogaster is a valuable model to study multiple aspects of normal animal physiology, stress response and disease. In this review, we discuss the contribution of Drosophila studies to the current knowledge on p53 and highlight recent evidences pointing to p53 novel roles in promoting tissue homeostasis and metabolic adaptation.

MicroRNA-mediated regulation of Dp53 in the Drosophila fat body contributes to metabolic adaptation to nutrient deprivation.

Multiple conserved mechanisms sense nutritional conditions and coordinate metabolic changes in the whole organism. We unravel a role for the Drosophila homolog of p53 (Dp53) in the fat body (FB; a functional analog of vertebrate adipose and hepatic tissues) in starvation adaptation. Under nutrient deprivation, FB-specific depletion of Dp53 accelerates consumption of major energy stores and reduces survival rates of adult flies. We show that Dp53 is regulated by the microRNA (miRNA) machinery and miR-305 in a nutrition-dependent manner. In well-fed animals, TOR signaling contributes to miR-305-mediated inhibition of Dp53. Nutrient deprivation reduces the levels of miRNA machinery components and leads to Dp53 derepression. Our results uncover an organism-wide role for Dp53 in nutrient sensing and metabolic adaptation and open up avenues toward understanding the molecular mechanisms underlying p53 activation under nutrient deprivation.

Aneuploidy and tumorigenesis in Drosophila.

Aneuploidy, described as an abnormal number of whole chromosomes or parts of them, has been observed in the majority of sporadic carcinomas, the most common type of cancer occurring in humans and derived from putative epithelial cells. However, the causal relationship between aneuploidy and tumorigenesis remains highly debated. On the one hand, aneuploidy has been shown to be a powerful driver of tumor progression, anticancer drug resistance, and tumor relapse. On the other hand, aneuploidy causes proteotoxic and metabolic stress, which compromises cell cycle proliferation and growth. Here we discuss the role of aneuploidy in tumorigenesis in light of the contribution of Drosophila epithelial cancer models and propose a stress-induced tumor-promoting role of aneuploidy.

Aneuploidy, cell delamination and tumorigenesis in Drosophila epithelia.

Chromosomal instability (CIN) is a common feature in human cancer, and highly aneuploid tumors are frequently associated with poor prognosis; however, the molecular and cellular mechanisms underlying CIN-induced tumorigenesis are poorly understood. Here we review recent findings about the role of CIN in driving tumor-like growth and host invasiveness in Drosophila epithelia and discuss the commonalities of CIN-induced tumors with other Drosophila-based cancer models. We also discuss possible scenarios that can account for the participation of CIN in tumorigenesis and propose that, alternatively to the classical role of aneuploidy in promoting the accumulation of mutations in cancer cells, aneuploidy can be a source of stress that may contribute to cancer initiation and/or progression.

Aneuploidy-induced delaminating cells drive tumorigenesis in Drosophila epithelia.

Genomic instability has been observed in essentially all sporadic carcinomas. Here we use Drosophila epithelial cells to address the role of chromosomal instability in cancer development as they have proved useful for elucidating the molecular mechanisms underlying tumorigenic growth. We first show that chromosomal instability leads to an apoptotic response. Interestingly, this response is p53 independent, as opposed to mammalian cells, and depends on the activation of the c-Jun N-terminal kinase (JNK) signaling cascade. When prevented from undergoing programmed cell death (PCD), chromosomal instability induces neoplasic overgrowth. These tumor-like tissues are able to grow extensively and metastasize when transplanted into the abdomen of adult hosts. Detailed analysis of the tumors allows us to identify a delaminating cell population as the critical one in driving tumorigenesis. Cells loose their apical-basal polarity, mislocalize DE-cadherin, and delaminate from the main epithelium. A JNK-dependent transcriptional program is activated specifically in delaminating cells and drives nonautonomous tissue overgrowth, basement membrane degradation, and invasiveness. These findings unravel a general and rapid tumorigenic potential of genomic instability, as opposed to its proposed role as a source of mutability to select specific tumor-prone aneuploid cells, and open unique avenues toward the understanding of the role of genomic instability in human cancer.

The interplay between morphogens and tissue growth.

Morphogens are conserved, secreted signalling molecules that regulate the size, shape and patterning of animal tissues and organs. Recent experimental evidence has emphasized the fundamental role of tissue growth in expanding the expression domains of morphogens and their target genes, in generating morphogen gradients and in modulating the response of cells to morphogens. Moreover, the classic view of how morphogens, particularly through their concentration gradient, regulate tissue size during development has been revisited recently. In this review, we discuss how morphogens and tissue growth affect each other, and we attempt to integrate genetic and molecular evidence from vertebrate and invertebrate model systems to put forward the idea that the interaction between growth and morphogens is a general feature of highly proliferative tissues.

A dp53-dependent mechanism involved in coordinating tissue growth in Drosophila.

Coordination of growth between and within organs contributes to the generation of well-proportioned organs and functionally integrated adults. The mechanisms that help to coordinate the growth between different organs start to be unravelled. However, whether an organ is able to respond in a coordinated manner to local variations in growth caused by developmental or environmental stress and the nature of the underlying molecular mechanisms that contribute to generating well-proportioned adult organs under these circumstances remain largely unknown. By reducing the growth rates of defined territories in the developing wing primordium of Drosophila, we present evidence that the tissue responds as a whole and the adjacent cell populations decrease their growth and proliferation rates. This non-autonomous response occurs independently of where growth is affected, and it is functional all throughout development and contributes to generate well-proportioned adult structures. Strikingly, we underscore a central role of Drosophila p53 (dp53) and the apoptotic machinery in these processes. While activation of dp53 in the growth-depleted territory mediates the non-autonomous regulation of growth and proliferation rates, effector caspases have a unique role, downstream of dp53, in reducing proliferation rates in adjacent cell populations. These new findings indicate the existence of a stress response mechanism involved in the coordination of tissue growth between adjacent cell populations and that tissue size and cell cycle proliferation can be uncoupled and are independently and non-autonomously regulated by dp53.

Oxygen sensing in Drosophila: multiple isoforms of the prolyl hydroxylase fatiga have different capacity to regulate HIFalpha/Sima.

BACKGROUND: The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia. Three HIF-prolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. METHODOLOGY AND PRINCIPAL FINDINGS: We have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. CONCLUSIONS AND SIGNIFICANCE: Diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain.

Drosophila genome-wide RNAi screen identifies multiple regulators of HIF-dependent transcription in hypoxia.

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke.

Central role of the oxygen-dependent degradation domain of Drosophila HIFalpha/Sima in oxygen-dependent nuclear export.

The Drosophila HIFalpha homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that nuclear import depends on an atypical bipartite nuclear localization signal mapping next to the C-terminus of the protein. We show here that nuclear export is mediated in part by a CRM1-dependent nuclear export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent nuclear export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid nuclear export of Sima occurs, whereas in hypoxia, Sima nuclear export is largely inhibited. HIFalpha/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between nuclear import and nuclear export, and nuclear export is modulated by oxygen tension.

Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting.

Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. Here, we show that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-alpha homolog Sima and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. We propose that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting.

Cellular and developmental adaptations to hypoxia: a Drosophila perspective.

The fruit fly Drosophila melanogaster, a widely utilized genetic model, is highly resistant to oxygen starvation and is beginning to be used for studying physiological, developmental, and cellular adaptations to hypoxia. The Drosophila respiratory (tracheal) system has features in common with the mammalian circulatory system so that an angiogenesis-like response occurs upon exposure of Drosophila larvae to hypoxia. A hypoxia-responsive system homologous to mammalian hypoxia-inducible factor (HIF) has been described in the fruit fly, where Fatiga is a Drosophila oxygen-dependent HIF prolyl hydroxylase, and the basic helix-loop-helix Per/ARNT/Sim (bHLH-PAS) proteins Sima and Tango are, respectively, the Drosophila homologues of mammalian HIF-alpha (alpha) and HIF-beta (beta). Tango is constitutively expressed regardless of oxygen tension and, like in mammalian cells, Sima is controlled at the level of protein degradation and subcellular localization. Sima is critically required for development in hypoxia, but, unlike mammalian model systems, it is dispensable for development in normoxia. In contrast, fatiga mutant alleles are all lethal; however, strikingly, viability to adulthood is restored in fatiga sima double mutants, although these double mutants are not entirely normal, suggesting that Fatiga has Sima-independent functions in fly development. Studies in cell culture and in vivo have revealed that Sima is activated by the insulin receptor (InR) and target-of-rapamycin (TOR) pathways. Paradoxically, Sima is a negative regulator of growth. This suggests that Sima is engaged in a negative feedback loop that limits growth upon stimulation of InR/TOR pathways.

Differential involvement of ERK1-2 and p38MAPK activation on Swiss 3T3 cell proliferation induced by prostaglandin F2alpha.

Prostaglandin F2alpha (PGF2alpha) induces cyclin D1 expression and DNA synthesis in Swiss 3T3 cells. In order to assess which signaling mechanisms are implicated in these processes, we have used both a pharmacological approach and interfering mutants. We demonstrate that PGF2alpha induces extracellular-signal-regulated kinase (ERK1-2) and p38MAPK activation, and inhibition of any of these signaling pathways completely blocks PGF2alpha-stimulated DNA synthesis. We also show that ERK1-2, but not p38MAPK activation is required to induce cyclin D1 expression, strongly suggesting that the concerted action of cyclin D1 gene expression and other events are required to induce complete phosphorylation of retinoblastoma protein and S-phase entry in response to PGF2alpha.

Leukemia inhibitory factor induces DNA synthesis in Swiss mouse 3T3 cells independently of cyclin D1 expression through a mechanism involving MEK/ERK1/2 activation.

Leukemia inhibitory factor (LIF) and oncostatin M (OSM) induce DNA synthesis in Swiss 3T3 cells through common signaling mechanism(s), whereas other related cytokines such as interleukin-6 and ciliary neurotrophic factor do not cause this response. Induction of DNA replication by LIF or prostaglandin F2alpha (PGF2alpha) occurs, in part, through different signaling events. LIF and OSM specifically trigger STAT1 cytoplasmic to nuclear translocation, whereas PGF2alpha fails to do so. However, LIF and PGF2alpha can trigger increases in ERK1/2 activity, which are required for their mitogenic responses because U0126, a MEK1/2 inhibitor, prevents both ERK1/2 activation and induction of DNA synthesis by LIF or PGF2alpha treatment. PGF2alpha induces cyclin D expression and full phosphorylation of retinoblastoma protein. In contrast, LIF fails to promote increases in cyclin D mRNA/protein levels; consequently, LIF induces DNA synthesis without promoting full phosphorylation of retinoblastoma protein (Rb). However, both LIF and PGF2alpha increase cyclin E expression. Furthermore, LIF mitogenic action does not involve protein kinase C (PKC) activation, because a PKC inhibitor does not block this effect. In contrast, PKC activity is required for PGF2alpha mitogenic action. More importantly, the synergistic effect between LIF and PGF2alpha to promote S phase entry is independent of PKC activation. These results show fundamental differences between LIF- and PGF2alpha-dependent mechanism(s) that induce cellular entry into S phase. These findings are critical in understanding how LIF and other related cytokine-regulated events participate in normal cell cycle control and may also provide clues to unravel crucial processes underlying cancerous cell division.

The insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-alpha/Sima.

The hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of a constitutively expressed HIF-beta subunit and an oxygen-regulated HIF-alpha subunit. We have previously defined a hypoxia-inducible transcriptional response in Drosophila melanogaster that is homologous to the mammalian HIF-dependent response. In Drosophila, the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) are the functional homologues of the mammalian HIF-alpha and HIF-beta subunits, respectively. HIF-alpha/Sima is regulated by oxygen at several different levels that include protein stability and subcellular localization. We show here for the first time that insulin can activate HIF-dependent transcription, both in Drosophila S2 cells and in living Drosophila embryos. Using a pharmacological approach as well as RNA interference, we determined that the effect of insulin on HIF-dependent transcriptional induction is mediated by PI3K-AKT and TOR pathways. We demonstrate that stimulation of the transcriptional response involves upregulation of Sima protein but not sima mRNA. Finally, we have analyzed in vivo the effect of the activation of the PI3K-AKT pathway on the subcellular localization of Sima protein. Overexpression of dAKT and dPDK1 in normoxic embryos provoked a major increase in Sima nuclear localization, mimicking the effect of a hypoxic treatment. A similar increase in Sima nuclear localization was observed in dPTEN homozygous mutant embryos, confirming that activation of the PI3K-AKT pathway promotes nuclear accumulation of Sima protein. We conclude that regulation of HIF-alpha/Sima by the PI3K-AKT-TOR pathway is a major conserved mode of regulation of the HIF-dependent transcriptional response in Drosophila.

The diagnostic performance of recombinant Trypanosoma cruzi ribosomal P2beta protein is influenced by its expression system.

In the present work, we have determined the effect of expression vectors and their corresponding host bacteria on the antigenic performance of Trypanosoma cruzi P2beta (TcP2beta) full-length recombinant protein. The gene encoding the TcP2beta ribosomal protein was cloned in pMAL-c2 and pET-32a vectors that allow the expression of high levels of soluble fusion proteins. A panel of 32 positive and 32 negative sera was assayed with the purified proteins expressed using pMal-c2 (TcP2beta-MBP) and pET-32a (TcP2beta-TRX) vectors and with MBP and TRX purified from pMAL-c2 and pET-32a vectors, respectively. The antigenic behavior of each TcP2beta recombinant protein differed in the diagnostic performance in terms of DI(+) (93.7 for TcP2beta-MBP vs 100% for TcP2beta-TRX), in DI(-) (90.5 for TcP2beta-MBP vs 100% for TcP2beta-TRX) and in cross-reaction with negative sera. To determine if the higher reactivity of expressed pMAL-c2 protein was due to folding during protein expression or to a steric effect related to the protein adsorption at the titration plate, the reactivity of sera against soluble proteins was assessed by ELISA inhibition assays. As each soluble protein preserved its level of reactivity, we concluded that differences in reactivity were due to intrinsic characteristics of the proteins and not to differences in patterns of adsorption to the plates.