InteractiveFly: GeneBrief

abnormal wing discs: Biological Overview | References


Gene name - abnormal wing discs

Synonyms -

Cytological map position - 3-102

Function - enzyme

Keywords - functions in endocytosis - required for proper internalization of receptors controlling different signaling pathways - dynamin-dependent vesicle recycling - microtubule-associated nucleoside diphosphate kinase that converts GDP to GTP - tumor suppressor

Symbol - awd

FlyBase ID: FBgn0000150

Genetic map position - chr3R:31,745,172-31,746,077

NCBI classification - Nucleoside diphosphate kinase

Cellular location - cytoplasmic



NCBI links: EntrezGene, Nucleotide, Protein
Recent literature
Gil-Ranedo, J., Gonzaga, E., Jaworek, K. J., Berger, C., Bossing, T. and Barros, C. S. (2019). STRIPAK Members Orchestrate Hippo and Insulin Receptor Signaling to Promote Neural Stem Cell Reactivation. Cell Rep 27(10): 2921-2933.e2925. PubMed ID: 31167138
Summary:
Adult stem cells reactivate from quiescence to maintain tissue homeostasis and in response to injury. How the underlying regulatory signals are integrated is largely unknown. Drosophila neural stem cells (NSCs) also leave quiescence to generate adult neurons and glia, a process that is dependent on Hippo signaling inhibition and activation of the insulin-like receptor (InR)/PI3K/Akt cascade. A transcriptome analysis was performed of individual quiescent and reactivating NSCs harvested directly from Drosophila brains; and the conserved STRIPAK complex members mob4, cka, and PP2A (microtubule star, mts) were identified. PP2A/Mts phosphatase, with its regulatory subunit Widerborst, maintains NSC quiescence, preventing premature activation of InR/PI3K/Akt signaling. Conversely, an increase in Mob4 and Cka levels promotes NSC reactivation. Mob4 and Cka are essential to recruit PP2A/Mts into a complex with Hippo kinase, resulting in Hippo pathway inhibition. It is proposed that Mob4/Cka/Mts functions as an intrinsic molecular switch coordinating Hippo and InR/PI3K/Akt pathways and enabling NSC reactivation.
BIOLOGICAL OVERVIEW

The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/NME2 metastasis suppressor genes. Awd acts in multiple tissues where its function is critical in establishing and maintaining epithelial integrity. This study analysed awd gene function in Drosophila epithelial cells using transgene-mediated RNA interference and genetic mosaic analysis. awd knockdown in larval wing disc epithelium leads to chromosomal instability (CIN) and induces apoptosis mediated by activation of c-Jun N-terminal kinase. Forced maintenance of Awd depleted cells, by expressing the cell death inhibitor p35, downregulates atypical protein kinase C and DE-Cadherin. Consistent with their loss of cell polarity and enhanced level of matrix metalloproteinase 1, cells delaminate from wing disc epithelium. Furthermore, the DNA content profile of these cells indicates that they are aneuploid. Overall, these data demonstrate a novel function for awd in maintenance of genomic stability. These results are consistent with other studies reporting that NME1 down-regulation induces CIN in human cell lines and suggest that Drosophila model could be successfully used to study in vivo the impact of NME/Awd induced genomic instability on tumour development and metastasis formation (Romani, 2017).

Genomic stability is critical for cell survival and development and several cellular mechanisms act to maintain genomic integrity. Failure of these mechanisms underlies aging and can lead to malignancies such as cancer and age-related neurodegenerative diseases. Chromosomal instability (CIN) is a form of genomic instability that often leads to aneuploidy, a deleterious condition characterised by copy number changes affecting part or whole chromosomes. Several dysfunctions could lead to CIN. Defective activity of the spindle assembly checkpoint (SAC), a signalling pathway that blocks anaphase onset in response to mis-attachment of chromosomes to the mitotic spindle, leads to CIN and aneuploidy. Work in Drosophila showed that loss of function of SAC genes as well as loss of function of genes involved in spindle assembly, chromatin condensation and cytokinesis induce CIN. More recent work in larval disc epithelia has shown that down-regulation of these genes causes apoptotic cell death trough activation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, blocking CIN-induced apoptotic cell death induces tumourigenic behaviour including basement membrane degradation, cell delamination, tissue overgrowth and aneuploidy (Romani, 2017).

The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/2 metastasis suppressor genes. Awd is a well-known endocytic mediator whose function is required in multiple tissues during development. Genetic studies showed that Awd endocytic function ensures appropriate internalisation of chemotactic signalling receptors such as platelet-derived growth factor/VEGF receptor (PVR) and fibroblast growth factor receptor (FGFR) and thus it regulates invasion and cellular motility. Furthermore, this endocytic function regulates Notch receptor trafficking and is required for maintenance of epithelial integrity as it controls the turnover of adherens junction components in ovarian somatic follicle cells. Consistent with the high degree of functional conservation between Awd and its mammalian counterparts, recent studies have shown a role for the NME1/2 proteins in vesicular transport (Romani, 2017).

This work has extended an analysis of the functional conservation between Awd and NME1/2 proteins. Since loss of NME1 gene function in human cell culture leads to polyploidy, this study have explored the role of Awd in maintenance of genomic stability. The data show that knockdown of awd in wing disc cells leads to CIN and to the CIN-induced biological responses mediated through JNK activation. Furthermore, when combined with block of apoptosis, down-regulation of awd leads to cell delamination and aneuploidy. Thus, the results of this in vivo analysis show a novel function for awd in maintenance of genomic stability (Romani, 2017).

Depletion of Awd triggers JNK-mediated cell death of wing disc cells and blocking the cell death machinery results in aneuploidy and cell delamination without overt hyperproliferative effect. Overgrowth of wing disc hosting aneuploid cells is due to activation of the JNK pathway that promotes expression of Wingless (Wg) upon blocking of apoptotic cell death. Wg is a mitogenic molecule required in the imaginal discs for growth and patterning and its expression in the aneuploid, delaminating CIN cells triggers growth of neighbouring non-delaminating cells. However, awd J2A4 mutant wing disc cells do not express Wg as a consequence of faulty Notch signalling; therefore, these cells cannot promote hyperplasia of the surrounding tissue. Furthermore, lack of hyperproliferation is also observed when an aneuploid condition arises from impaired activity of genes controlling karyokinesis. The diaphanous gene (dia) codes for an actin-regulatory molecule that is required during acto-myosin driven contraction of metaphase furrows. Simultaneous depletion of dia gene expression and blocking of apoptosis do not lead to hyperplastic growth probably due to defective karyokinesis. Intriguingly, Awd is a microtubule-associated nucleoside diphosphate kinase that converts GDP to GTP and the analysis of awd mutant larval brain showed mitotic defects correlated with defective microtubule polymerisation. This raises the possibility that the Awd kinase function plays a role in GTP supply to proteins such as Orbit that are required for stabilisation of spindle microtubules (Romani, 2017).

Two lines of evidence further support the hypothesis that Awd could be involved in karyokinesis. The first comes from studies showing that endosome trafficking and transport to the intercellular bridges of dividing cells plays a critical role during abscission, the last step of karyokinesis. In addition, remodelling the of plasma membrane that underlies nuclear divisions in the syncytial embryo and cellularisation also requires endocytosis. Embryonic cellularisation requires the dynamin encoded by the shibire (shi) locus and Rab5 GTPase function, since loss of function of either genes arrests ingression of metaphase furrows. Awd functionally interacts with shi locus and Awd is also required for Rab5 function in early endosomes. Thus, a possible role for Awd in cytokinesis should be considered (Romani, 2017).

The second line of evidence comes from studies on NME1, the human homolog of awd gene. This metastasis suppressor gene shares about 78% of amino acid identity with the awd gene. Down-regulation of NME1 gene expression in diploid cells results in cytokinesis failure and leads to tetraploidy. The in vivo results show that Awd plays a role in maintenance of genomic stability, confirming the high degree of conservation between NME1 and Awd proteins. Drosophila studies have already been crucial in identification of NME1 function in epithelial morphogenesis, and the present work shows that this function can be a useful model for impact on tumour development and progression (Romani, 2017).

Notch signaling during development requires the function of awd, the Drosophila homolog of human metastasis suppressor gene Nm23

The Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. The cellular function of the mammalian members of this family, the Nm23 proteins, has not yet been clearly defined. Previous awd genetic analyses unraveled its endocytic role that is required for proper internalization of receptors controlling different signaling pathways. This study analyzed the role of Awd in controlling Notch signaling during development. To study the awd gene function, genetic mosaic approaches were used to obtain cells homozygous for a loss of function allele. In awd mutant follicle cells and wing disc cells, Notch accumulates in enlarged early endosomes, resulting in defective Notch signaling. The results demonstrate that awd function is required before γ-secretase mediated cleavage since over-expression of the constitutively active form of the Notch receptor in awd mutant follicle cells allows rescue of the signaling. By using markers of different endosomal compartments it was shown that Notch receptor accumulates in early endosome in awd mutant follicle cells. Trafficking assay in living wing discs also shows that Notch accumulates in early endosomes. Importantly, constitutively active Rab5 cannot rescue the awd phenotype, suggesting that awd is required for Rab5 function in early endosome maturation. This report has demonstrated that awd is essential for Notch signaling via its endocytic role. In addition this study has identified the endocytic step at which Awd function is required for Notch signaling and evidence was obtained indicating that Awd is necessary for Rab5 function. These findings provide new insights into the developmental and pathophysiological function of this important gene family (Ignesti, 2014).

This report demonstrates a role of awd in regulating Notch signaling via its endocytic function including surface internalization and vesicle trafficking. This conclusion is based on results that show: (1) multiple Notch target genes are mis-expressed in follicle cells and wing discs, (2) Notch accumulates in enlarged early endosomes, and (3) awd function is required for the Rab5 activity in early endosome maturation. The results also indicate that during vesicles trafficking, the Awd action is downstream of the S2 cleavage, since over-expression of NEXT (Notch EXternal Truncation) accumulated intracellularly and could not rescue the awd defect. The same NEXT over-expression strategy could rescue the shi/dynamin defect, strongly supporting the notion that the Awd action on Notch signaling is post-membrane invagination. Since over-expression of NICD could rescue the awd defect, the Awd action is likely upstream or in parallel to the S3 cleavage event (γ-secretase activity). Although a role of awd in promoting the activity of γ-secretase cannot be completely ruled out, this possibility is considered unlikely. First, awd is a known endocytic factor demonstrated in multiple tissues including neurons, trachea, and follicle cells. Second, neither the expression level nor the expression pattern of Presenilin, the catalytic subunit of γ-secretase, is altered in awd mutant cells. Third, if the defect is in γ-secretase function, it would be expected that Notch should accumulate in Hrs-positive MVBs. On the contrary, such ectopic accumulation of Notch was not observed in Hrs-positive vesicles. Therefore, the results, in aggregate, suggest that the main action of Awd on Notch signaling is via its endocytic activity promoting the transition from early endosomes to late endosomes. However, potential defects downstream of γ-secretase cleavage, such as trafficking to nucleus, in awd mutant cannot be formally ruled out (Ignesti, 2014).

One curious exception for the awd function in relation to Notch signaling is found in the border cells. During the migration of these cells, Awd expression is down-regulated. Re-expression of Awd can lead to reduction of surface receptors, such as PVR that is critical for directional movement, resulting in defective migration. Interestingly, Notch signaling is also important for border cell migration. It therefore appears that Notch signaling in these specialized cells does not require Awd activity or is insensitive to Awd protein levels. To test this, Notch expression in border cells was compared with or without Awd re-expression. In wild-type border cells (no Awd), Notch is located on the cell surface as well as in the cell body, consistent with active signaling. Forced re-expression of Awd in the border cells does not alter this pattern. This may be because Notch is already actively internalized; increasing the Awd level cannot further enhance such activity. Indeed, endocytosis is intrinsically highly active in border cells. Alternatively, the differential dependence of Notch on Awd activity may be a function of how Notch is activated, not how Awd functions differently in different cell types. For example, it has been shown that the Notch ligand Delta may be co-expressed with Notch in the same border cells. Recent reports have hinted that the requirement of endocytosis for Notch signaling may depend on the ligand-receptor relationship (for example, ligand-dependent or -independent, trans- or cis-activation, and so on). It is therefore considered that the apparent Awd-independent Notch signaling in border cells has more to do with the intrinsic Notch signaling mechanism in these cells, and less to do with the function of Awd (Ignesti, 2014).

The results indicate that the Notch signaling defect in awd mutant cells is the failure to deliver Notch past the Rab5-dependent early endosomal stage. On the other hand, the ESCRT complex mutants, which are defective in late endosome formation, promote Notch signaling. Taken together, it appears that Notch activation occurs in the intermediate stage between early endosome formation and late endosome entry. Transition from early endosomes to late endosomes is accompanied by cargo sorting, intravesicular invagination and acidification of the luminal contents. Curiously, the matured early endosome and MVB marker hrs mutant has no effect on Notch signaling, which indicates that endosomal cargo sorting per se is not required for Notch signaling. It was also shown that awd mutant cells do not exhibit altered levels of Lysotracker staining and that endosomal Notch remains on the surface of enlarged endosomes in awd mutants. The exact nature of this transition state that favors Notch processing, therefore, requires further analysis. The endocytic function of awd has traditionally been described as a 'GTP supplier' for Dynamin, based on genetic interaction data and logical extrapolation because of the GTP producing activity of Awd. This report demonstrates that, in relation to Notch signaling, awd functions downstream of, but not directly on, dynamin. It is instead critical for Rab5 activity. This is supported by the following evidence: 1) Notch in awd mutant accumulates in Avl-containing vesicles. Therefore, the awd defect is post Dynamin-mediated cleavage of membrane invagination. 2) Rab5CA can push Notch into enlarged early endosomes but failed to rescue the awd phenotype, thereby strengthening the notion that awd defect is post Shi/Dynamin function. 3) The Notch accumulation pattern in shi mutant is different from that in awd mutant. 4) Over-expression of NEXT could not rescue awd defect. The same NEXT over-expression strategy could rescue the shi defect, strongly supporting the notion that the Awd action concerning Notch signaling is post-membrane invagination. It should be noted that surface accumulation was observed of NECD antibody-detected Notch molecules, likely representing the full-length Notch not engaged in ligand binding and signaling. This indicates that Awd can affect constitutive internalization of full-length Notch (Ignesti, 2014).

The requirement of endocytosis in the signal-receiving cells for Notch activation has been amply demonstrate. It has been shown that Notch signaling in follicle cells after stage 6 requires Delta. Since this report showed that Notch signaling cannot occur in the follicle cell without awd function, it is concluded that, at least in follicle cells, endocytosis is a requisite process for ligand-dependent Notch signaling (Ignesti, 2014).

The involvement of endocytosis in Notch signaling is significant since many of the endocytic components shown to regulate Notch signaling have also been implicated in carcinogenesis. For example, V-ATPase is required for Notch signaling while mutations in ESCRT components, such as Tsg101, result in increased Notch signaling. V-ATPase has generally been considered an oncogene because it is associated with acidification of tumor cells. ESCRT components, on the other hand, have been shown to suppress tumor formation because they down-regulate surface growth factor receptor signaling. As such, attempts to design therapeutics based on these prevalent functions should take into account the effects on Notch signaling, since the relationship between Notch signaling and carcinogenesis is context-dependent (Ignesti, 2014).

Awd belongs to the Nm23 family of protein that is evolutionarily conserved from Drosophila to mammals. This in vivo analyses has demonstrated that loss of awd gene function blocks Notch signaling by altering the receptor processing after the S2 cleavage and causes Notch accumulation in early endosomes. Furthermore, evidence was obtained indicating that Awd is required for Rab5 function in early endosome formation (Ignesti, 2014).

Nm23 has been an enigmatic gene function. It is a housekeeping gene involved in nucleotide synthesis and energy metabolism, and yet exhibiting specific developmental functions. It was the first metastasis suppressor gene identified, yet exhibits oncogenic functions in some cancer cohorts. Previous work has shown that either loss-of-function or over-expression of awd can affect different aspects of epithelial morphogenesis. That is, loss-of-function awd results in over-accumulation of adherens junction components and piling up of the epithelium, while over-expression of awd results in reduced adherens junctions and disintegration of epithelial structure. These findings provided some explanation of the biphasic function of Nm23 in tumorigenesis. In light of the current studies, an additional level of complexity should be considered since Notch signaling can exert different cellular functions in different tissues and at different times during pathophysiological alterations of the same tissues (Ignesti, 2014).

Dynamin controls extracellular level of Awd/Nme1 metastasis suppressor protein
Dynamin GTPase (Dyn) plays a critical role in membrane-remodelling events underlying endocytosis. Studies in Drosophila identified a functional interaction between the Dyn homologue, encoded by the shibire (shi) gene, and Abnormal wing discs (Awd), a nucleoside diphosphate kinase (NDPK) that is the homologue of group I Nme human genes. These Drosophila studies showed that awd mutations enhance mutant shi phenotype and thus indicated the existence of a highly specific interaction between these genes. Furthermore, in human cells, it has been shown that Nme proteins promote Dyn activity in different membrane compartments through spatially controlled supply of GTP. Interestingly, Awd and Nme proteins have been detected in the extracellular environment. While no role has been inferred for extracellular Awd, presence of Nme1 in cancer patient serum is an unfavourable prognostic marker. The present work used Drosophila and human cell line models to investigate the shuttling Awd/Nme1 proteins between intracellular and extracellular spaces. By using classic and reverse genetic approaches, downregulation of Shi/Dyn1 activity was shown to enhance extracellular Awd/Nme1 in both Drosophila and human colon cell lines. This analysis was extended to colon cancer cell lines, and knocking down Dyn1, in addition to raising Nme1 extracellular amount, was shown to downregulates expression of molecular components that play key roles in tumour invasion. Interestingly, in vivo analyses of Drosophila larval adipocytes showed that the conditional block of Shi activity greatly reduces intracellular amounts of Awd confirming that Shi plays a key role in controlling the balance between intracellular and extracellular Awd (Romani, 2016).

miR-2 family targets awd and fng to regulate wing morphogenesis in Bombyx mori

MicroRNAs (miRNAs) are post-transcriptional regulators that target specific mRNAs for repression and thus play key roles in many biological processes, including insect wing morphogenesis. miR-2 is an invertebrate-specific miRNA family that has been predicted in the fruit fly, Drosophila melanogaster, to be involved in regulating the Notch signaling pathway. This study shows that miR-2 plays a critical role in wing morphogenesis in the silkworm, Bombyx mori, a lepidopteran model insect. Transgenic over-expression of a miR-2 cluster using a Gal4/UAS system results in deformed adult wings, supporting the conclusion that miR-2 regulates functions essential for normal wing morphogenesis. Two genes, abnormal wing disc (awd) and fringe (fng), which are positive regulators in Notch signaling, are identified as miR-2 targets and validated by a dual-luciferase reporter assay. The relative abundance of both awd and fng expression products was reduced significantly in transgenic animals, implicating them in the abnormal wing phenotype. Furthermore, somatic mutagenesis analysis of awd and fng using the CRISPR/Cas9 system and knock-out mutants also resulted in deformed wings similar to those observed in the miR-2 over-expression transgenic animals. The critical role of miR-2 in Bombyx wing morphogenesis may provide a potential target in future lepidopteran pest control (Ling, 2015).

Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis

While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, interesting and unexpected mutant phenotypes were noticed in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch (Pu) and pale (ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up (Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. Expression of Punch, Ple, Catsup and dopamine was seen in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally it was shown that dopamine regulates endocytosis via controlling the dynamin protein level (Hsouna, 2007).

The data demonstrate that DA regulates FGF receptor turnover. Btl;;GFP fusion protein is down-regulated in the presence of DA and up-regulated in the presence of 3-IT, either in trachea or in cultured S2 cells. These results are consistent with the model that DA promotes internalization of Btl/FGFR, leading to its degradation through the endocytic pathway. The role of DA in internalization of FGFR is further suggested by the genetic interaction between the DA synthesis and endocytic pathway genes. Mutations in the human tumor suppressor nucleoside diphosphate kinase (NDK) gene (nm23) are strongly associated with tumor metastatic activity. Its functional homolog in Drosophila, abnormal wing discs (awd), has been shown to genetically interact with a temperature-sensitive allele of the shibire gene (shits), which encodes dynamin, a large GTPase required for the formation of clathrin-coated endocytic vesicles. A function for awd/nm23 in the migratory phase of tracheal development has been demonstrated and functional interactions occur with shi/dynamin that are required for the modulation of FGFR/Btl levels in tracheal cells. This report shows that Pu and ple phenotypes are exacerbated by awd and rescued by btl, while the Catsup phenotypes are rescued by awd but exacerbated by btl. These results indicate that Pu/GTPCH and Ple/TH, and by extension, DA, are positive regulators of endocytosis (Hsouna, 2007).

The Drosophila metastasis suppressor gene Nm23 homolog, awd, regulates epithelial integrity during oogenesis

The expression levels of the metastasis suppressor gene Nm23 have been shown to correlate either positively or inversely with prognosis in different cancer cohorts. This indicates that Nm23 may be needed at different expression levels and may function differently in various tissues. This study reports a novel epithelial function of the Drosophila melanogaster homolog of human Nm23, abnormal wing discs (awd). A dynamic expression pattern is shown of the Awd protein during morphogenesis of the Drosophila follicle cells during oogenesis. Loss-of-function awd mutant cells result in the accumulation and spreading of adherens junction components, such as Drosophila E-cadherin, beta-catenin/Armadillo, and alpha-spectrin, and the disruption of epithelial integrity, including breaking up of the epithelial sheet and piling up of follicle cells. In contrast, overexpression of awd diminishes adherens junction components and induces a mesenchymal cell like cell shape change. The gain-of-function phenotype is consistent with a potential oncogenic function of this metastasis suppressor gene. Interestingly, this study demonstrated that the epithelial function of awd is mediated by Rab5 and showed that the Rab5 expression level is downregulated in awd mutant cells. Therefore, awd modulates the level and localization of adherens junction components via endocytosis. This is the first demonstration of an in vivo function of Nm23 family genes in regulating epithelial morphogenesis (Woolworth, 2009).

awd, the homolog of metastasis suppressor gene Nm23, regulates Drosophila epithelial cell invasion

Border cell migration during Drosophila oogenesis is a highly pliable model for studying epithelial to mesenchymal transition and directional cell migration. The process involves delamination of a group of 6 to 10 follicle cells from the epithelium followed by guided migration and invasion through the nurse cell complex toward the oocyte. The guidance cue is mainly provided by the homolog of platelet-derived growth factor/vascular endothelial growth factor family of growth factor, Pvf, emanating from the oocyte, although Drosophila epidermal growth factor receptor signaling also plays an auxiliary role. Earlier studies implicated a stringent control of the strength of Pvf-mediated signaling since both down-regulation of Pvf and overexpression of active Pvf receptor (Pvr) resulted in stalled border cell migration. This study shows that the metastasis suppressor gene homolog Nm23/awd (abnormal wing discs) is a negative regulator of border cell migration. Its down-regulation allows for optimal spatial signaling from two crucial pathways, Pvr and JAK/STAT. Its overexpression in the border cells results in stalled migration and can revert the phenotype of overexpressing constitutive Pvr or dominant-negative dynamin (Shibire or Shi). The functional relationship between awd and shi is highly specific and almost exclusive in the endocytic pathway. The functional relationship between Nm23/Awd and dynamin has prompted the suggestion that Nm23/Awd is a GTP supplier for dynamin, a GTPase. Nonetheless, the putative antimetastasis activity of Nm23/Awd has never been demonstrated in a physiologically relevant metastasis or epithelial to mesenchymal transition model. This is a rare example demonstrating the relevance of a metastasis suppressor gene function utilized in a developmental process involving cell invasion (Nallamothu, 2008).

This report describes a novel role of a negative regulator of directional migration in border cells. Specifically, the significance of developmentally regulated loss of Awd expression in border cells during their active migratory phase was studied. Ectopic expression of Awd effectively blocks border cell movement, suggesting that Awd is involved in modulating the directional movement of the border cell complex. Conversely, a high level of Awd expression was observed in the nonmigrating follicle cells, possibly promoting rapid turnover of surface receptors to prevent ectopic cell migration. Indeed, loss of awd in these cells and in premigratory border cells resulted in up-regulation of Pvr and stalling of border cells, consistent with the phenotype of overactive Pvr signaling reported previously due to overexpression of wild-type Pvr. The results show that the function of Awd is to promote down-regulation of the surface receptors such as Pvr and Dome, in collaboration with Shi/dynamin, thereby regulating the chemotactic signal strength. Although the function of Awd has been linked to dynamin (Dammai, 2003; Krishnan, 2001), this report is the first on the relevance of the Nm23/Awd antimetastasis function in an analogous developmental model. This study has demonstrated that border cell migration is stalled by both ectopic expression of Awd in the migrating cells and knockdown of Awd in premigrating cells, although through opposite consequences of reducing and increasing Pvr expression, respectively. This is consistent with the published finding that both loss of function and gain of function in Pvr signaling can disrupt border cell migration, due to loss of chemotactic signal or overwhelming signal, respectively. It was proposed and subsequently demonstrated by time-lapse microscopy that border cells that are oversaturated with Pvf signaling spin around without moving forward, consistent with the overborne, nondirectional chemotactic signaling response, while pvr loss-of-function border cells do not move. That is, although the phenotypic outcomes are the same, the cellular behaviors of the two genetic conditions are just the opposite (Nallamothu, 2008).

At this time, the cellular events that precisely down-regulate Awd expression in migrating border cells remain unknown. However, the observations suggest that the regulatory mechanism, besides potential transcriptional regulation, could at least in part be posttranscriptional. For example, the slbo-GAL4 driver can usually induce very high levels of ectopic expression, as evidenced by the expression of UAS-{lambda}pvr in this study. However, with UAS-awd (without the endogenous 3' untranslated region), it was possible to achieve at best a level equal to the endogenous one in nearby follicle cells and very often much lower (Nallamothu, 2008).

The histidine-dependent phosphotransferase activity of Nm23/Awd has functional correlation with the production and usage of GTP and the Awd-GTP link is worth noting since dynamin is a GTPase. In a classic study to identify components of eye color pathway, one peculiar, otherwise healthy mutant caused dominant lethality in the viable eye color prune null mutant background (Sturtevant, 1956). This dominant conditional lethal allele was named Killer-of-prune (K-pn) and turned out to be a missense mutant allele of awd. This is highly interesting because the Drosophila eye pigmentation is determined by pteridines, which are also a precursor of essential enzyme cofactors. The rate-limiting enzyme in pteridine biosynthesis is GTP cyclohydrolase, which uses substrate GTP to generate dihydroneopterin triphosphate. It was suggested that the Prune protein, which contains pyrophosphatase activity, stabilizes or promotes Nm23/Awd multimeric protein activity by channeling the phosphate. It is possible that Awd and Prune proteins together form a relay system for generating GTP. Therefore, the K-pn mutation of awd in the prune mutant background renders the phosphate transfer function of the Prune-Awd protein complex even less stable. Indeed, among the myriad of interacting proteins of Nm23 in mammalian cells, many are related directly or indirectly to the GTPases, such as Arf6, TIAM1 (a guanine exchange factor for Rac), Lbc (a guanine exchange factor for Rho), and Rad. Whether or not these GTPase-related functions hold true requires further in vivo investigation. Recently, the lysophosphatidic acid receptor EDG2 was found to be overexpressed in Nm23-H1 mutant metastatic breast cancer cells, which can account for the metastatic activity of this cell line. However, whether the up-regulation is a direct or downstream effect of Nm23 loss of function is not clear. It therefore remained to be determined whether the similar receptor down-regulation mechanism by Awd observed in this report is applicable to EDG2 regulation (Nallamothu, 2008).

It should be noted, however, that although the observed genetic interaction between awd and shi suggests that Awd may promote the endocytic activity of Shi/dynamin, it is formally possible that Awd may promote protein turnover that is downstream of the initial endocytic event. On this note, it is also worth considering other activities of Nm23/Awd. The results showed that substitution of the active-site histidine residue that is critical for the nucleoside diphosphate kinase activity could not stall border cell migration. This is consistent with previous finding that this residue is required for rescuing the enhancer of shi phenotype (Krishnan, 2001). Curiously, this residue is not required for suppressing the in vitro motility (assayed by Boyden chamber) of the metastatic breast cancer cells. However, the histidine substitutions employed in the two systems are different (phenylalanine in human versus alanine in fly). It is therefore difficult at this time to draw a direct comparison. In contrast, human mutants that affect the histidine-dependent protein kinase activity failed to suppress the in vitro motility of the cancer cells. So far, very few Nm23 protein kinase targets have been identified and none verified in physiological settings. Nonetheless, the protein kinase activity may be of specific functional significance since the range of targets is likely limited, so that specific pathways that contribute to metastasis may be identified more readily. The border cell migration model describe here should be used in future studies to test the functions of Nm23/Awd based on the above-mentioned human mutations (Nallamothu, 2008).

Drosophila awd, the homolog of human nm23, regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

Human nm23 has been implicated in suppression of metastasis in various cancers, but the underlying mechanism of such activity has not been fully understood. Using Drosophila tracheal system as a genetic model, this study examined the function of the Drosophila homolog of nm23, the abnormal wing disc (awd) gene, in cell migration. Loss of Drosophila awd results in dysregulated tracheal cell motility. This phenotype can be suppressed by reducing the dosage of the chemotactic FGF receptor (FGFR) homolog, breathless (btl), indicating that btl and awd are functionally antagonists. In addition, mutants of shi/dynamin show similar tracheal phenotypes as in awd and exacerbate those in awd mutant, suggesting defects in vesicle-mediated turnover of FGFR in the awd mutant. Consistent with this, Btl-GFP chimera expressed from a cognate btl promoter-driven system accumulate at high levels on tracheal cell membrane of awd mutants as well as in awd RNA duplex-treated cultured cells. Thus, it is proposed that awd regulates tracheal cell motility by modulating the FGFR levels, through a dynamin-mediated pathway (Dammai, 2003. Full text of article).

Nucleoside diphosphate kinase, a source of GTP, is required for dynamin-dependent synaptic vesicle recycling

Nucleoside diphosphate kinase (NDK), an enzyme encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, generates nucleoside triphosphates from respective diphosphates. This study demonstrated that NDK regulates synaptic vesicle internalization at the stage where function of the dynamin GTPase is required. awd mutations lower the temperature at which behavioral paralysis, synaptic failure, and blocked membrane internalization occur at dynamin-deficient, shits, mutant nerve terminals. Hypomorphic awd alleles display shits-like defects. NDK is present at synapses and its enzymatic activity is essential for normal presynaptic function. A model is suggested in which dynamin activity in nerve terminals is highly dependent on NDK-mediated supply of GTP. This connection between NDK and membrane internalization further strengthens an emerging hypothesis that endocytosis, probably of activated growth factor receptors, is an important tumor suppressor activity in vivo (Krishnan, 2001).

The expression of the Drosophila awd gene during normal development and in neoplastic brain tumors caused by lgl mutations

The abnormal wing discs (awd) gene of Drosophila is homologous to the nm23 gene of mammals, a gene whose expression is altered in metastatic tumors. Both awd and nm23 encode nucleoside diphosphate kinases (NDP kinases). The accumulation of AWD/NDP kinase has been examined during normal development by assaying enzyme activity in extracts. There is a nearly constant level of activity throughout larval and pupal development. The tissue-specific transcription of the awd gene was examined by RNA in situ hybridization and by reporter gene expression. In imaginal discs and brains there is no detectable awd gene expression until the beginning of the third larval instar, despite the constant level of enzyme activity measured in extracts of larvae and pupae. The most intense awd gene expression in imaginal discs and brains occurs after the end of larval development. awd gene expression was examined in neoplastic brain tumors caused by mutations in the lethal (2) giant larvae gene. In l(2)gl mutant brains, as in normal brains, awd gene expression begins during the third larval instar. No tumors form in brains from l(2)gl-;awd- double mutant larva, so awd gene expression is required for tumor formation and/or proliferation. There is more accumulation of AWD/NDP kinase in l(2)gl- mutant brains than there is in normal brains. Using an awd reporter gene, it has been shown that this is a consequence of an increased proportion of awd gene-expressing cells in mutant brains. Using the same awd reporter gene as a marker of donor cells, the invasiveness of l(2)gl-induced neuroblastomas has been confirmed (Timmons, 1993).


Functions of Abnormal wing disc orthologs in other species

Small molecule activator of Nm23/NDPK as an inhibitor of metastasis

Nm23-H1/NDPK-A is a tumor metastasis suppressor having NDP kinase (NDPK) activity. Nm23-H1 is positively associated with prolonged disease-free survival and good prognosis of cancer patients. Approaches to increasing the cellular levels of Nm23-H1 therefore have significance in the therapy of metastatic cancers. A small molecule, (+/-)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene (NMac1), activates Nm23. NMac1 directly binds to Nm23-H1 and increases its NDPK activity. Employing various NMac1 derivatives and hydrogen/deuterium mass spectrometry (HDX-MS), this study identified the pharmacophore and mode of action of NMac1. NMac1 binds to the C-terminal of Nm23-H1 and induces the NDPK activation through its allosteric conformational changes. NMac1-treated MDA-MB-231 breast cancer cells showed dramatic changes in morphology and actin-cytoskeletal organization following inhibition of Rac1 activation. NMac1 also suppressed invasion and migration in vitro, and metastasis in vivo, in a breast cancer mouse model. NMac1 as an activator of NDPK has potential as an anti-metastatic agent (Lee, 2018).

NDK-1, the homolog of NM23-H1/H2 regulates cell migration and apoptotic engulfment in C. elegans

Abnormal regulation of cell migration and altered rearrangement of cytoskeleton are characteristic of metastatic cells. The first described suppressor of metastatic processes is NM23-H1, which displays NDPK (nucleoside-diphosphate kinase) activity. To better understand the role of nm23 genes in cell migration, this study investigated the function of NDK-1, the sole Caenorhabditis elegans homolog of group I NDPKs in distal tip cell (DTC) migration. Dorsal phase of DTC migration is regulated by integrin mediated signaling. ndk-1 loss of function mutants show defects in this phase. Epistasis analysis using mutants of the alpha-integrin ina-1 and the downstream functioning motility-promoting signaling module (referred to as CED-10 pathway) placed NDK-1 downstream of CED-10/Rac. As DTC migration and engulfment of apoptotic corpses are analogous processes, both partially regulated by the CED-10 pathway, this study investigated defects of apoptosis in ndk-1 mutants. Embryos and germ cells defective for NDK-1 showed an accumulation of apoptotic cell corpses. Furthermore, NDK-1::GFP is expressed in gonadal sheath cells, specialized cells for engulfment and clearence of apoptotic corpses in germ line, which indicates a role for NDK-1 in apoptotic corpse removal. In addition to the CED-10 pathway, engulfment in the worm is also mediated by the CED-1 pathway. abl-1/Abl and abi-1/Abi, which function in parallel to both CED-10/CED-1 pathways, also regulate engulfment and DTC migration. ndk-1(-);abi-1(-) double mutant embryos display an additive phenotype (e. g. enhanced number of apoptotic corpses) which suggests that ndk-1 acts in parallel to abi-1. Corpse number in ndk-1(-);ced-10(-) double mutants, however, is similar to ced-10(-) single mutants, suggesting that ndk-1 acts downstream of ced-10 during engulfment. In addition, NDK-1 shows a genetic interaction with DYN-1/dynamin, a downstream component of the CED-1 pathway. In summary, it is proposed that NDK-1/NDPK might represent a converging point of CED-10 and CED-1 pathways in the process of cytoskeleton rearrangement (Fancsalszky, 2014).

The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans

The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs was study, and focus was placed on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and it is suggested that NDK-1 modulates KSR activity through direct physical interaction. This study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis (Masoudi, 2013).


REFERENCES

Search PubMed for articles about Drosophila Abnormal wing discs

Dammai, V., Adryan, B., Lavenburg, K. R. and Hsu, T. (2003). Drosophila awd, the homolog of human nm23, regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development. Genes Dev. 17(22): 2812-24. PubMed Citation: 14630942

Fancsalszky, L., Monostori, E., Farkas, Z., Pourkarimi, E., Masoudi, N., Hargitai, B., Bosnar, M. H., Dezeljin, M., Zsakai, A., Vellai, T., Mehta, A. and Takacs-Vellai, K. (2014). NDK-1, the homolog of NM23-H1/H2 regulates cell migration and apoptotic engulfment in C. elegans. PLoS One 9(3): e92687. PubMed ID: 24658123

Hsouna, A., Lawal, H. O., Izevbaye, I., Hsu, T. and O'Donnell, J. M. (2007). Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis. Dev Biol 308(1): 30-43. PubMed ID: 17585895

Ignesti, M., Barraco, M., Nallamothu, G., Woolworth, J. A., Duchi, S., Gargiulo, G., Cavaliere, V. and Hsu, T. (2014). Notch signaling during development requires the function of awd, the Drosophila homolog of human metastasis suppressor gene Nm23. BMC Biol 12: 12. PubMed ID: 24528630

Krishnan, K. S., Rikhy, R., Rao, S., Shivalkar, M., Mosko, M., Narayanan, R., Etter, P., Estes, P. S. and Ramaswami, M. (2001). Nucleoside diphosphate kinase, a source of GTP, is required for dynamin-dependent synaptic vesicle recycling. Neuron 30(1): 197-210. PubMed ID: 11343655

Lee, J. J., Kim, H. S., Lee, J. S., Park, J., Shin, S. C., Song, S., Lee, E., Choi, J. E., Suh, J. W., Lee, H., Kim, E. E., Seo, E. K., Shin, D. H., Lee, H. Y., Lee, H. Y. and Lee, K. J. (2018). Small molecule activator of Nm23/NDPK as an inhibitor of metastasis. Sci Rep 8(1): 10909. PubMed ID: 30026594

Ling, L., Ge, X., Li, Z., Zeng, B., Xu, J., Chen, X., Shang, P., James, A. A., Huang, Y. and Tan, A. (2015). MiR-2 family targets awd and fng to regulate wing morphogenesis in Bombyx mori. RNA Biol 12(7):742-8. PubMed ID: 26037405

Masoudi, N., Fancsalszky, L., Pourkarimi, E., Vellai, T., Alexa, A., Remenyi, A., Gartner, A., Mehta, A. and Takacs-Vellai, K. (2013). The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans. Development 140(16): 3486-3495. PubMed ID: 23900546

Nallamothu, G., Woolworth, J. A., Dammai, V. and Hsu, T. (2008). awd, the homolog of metastasis suppressor gene Nm23, regulates Drosophila epithelial cell invasion. Mol. Cell. Biol. 28: 1964-1973. PubMed Citation: 18212059

Romani, P., Papi, A., Ignesti, M., Soccolini, G., Hsu, T., Gargiulo, G., Spisni, E. and Cavaliere, V. (2016). Dynamin controls extracellular level of Awd/Nme1 metastasis suppressor protein. Naunyn Schmiedebergs Arch Pharmacol. PubMed ID: 27449069

Romani, P., Duchi, S., Gargiulo, G. and Cavaliere, V. (2017). Evidence for a novel function of Awd in maintenance of genomic stability. Sci Rep 7(1): 16820. PubMed ID: 29203880

Timmons, L., et al. (1993). The expression of the Drosophila awd gene during normal development and in neoplastic brain tumors caused by lgl mutations. Dev. Biol. 158(2): 364-79. PubMed Citation: 8393813

Woolworth, J. A., Nallamothu, G. and Hsu, T. (2009). The Drosophila metastasis suppressor gene Nm23 homolog, awd, regulates epithelial integrity during oogenesis. Mol Cell Biol 29(17): 4679-4690. PubMed ID: 19581292


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date revised: 10 May, 2019

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