The Drosophila ZNRF1/2 homologue, detour, interacts with HOPS complex and regulates autophagy.

Autophagy, the process of elimination of cellular components by lysosomal degradation, is essential for animal development and homeostasis. Using the autophagy-dependent Drosophila larval midgut degradation model we identified an autophagy regulator, the RING domain ubiquitin ligase CG14435 (detour). Depletion of detour resulted in increased early-stage autophagic vesicles, premature tissue contraction, and overexpression of detour or mammalian homologues, ZNRF1 and ZNRF2, increased autophagic vesicle size. The ablation of ZNRF1 or ZNRF2 in mammalian cells increased basal autophagy. We identified detour interacting proteins including HOPS subunits, deep orange (dor/VPS18), Vacuolar protein sorting 16A (VPS16A), and light (lt/VPS41) and found that detour promotes their ubiquitination. The detour mutant accumulated autophagy-related proteins in young adults, displayed premature ageing, impaired motor function, and activation of innate immunity. Collectively, our findings suggest a role for detour in autophagy, likely through regulation of HOPS complex, with implications for healthy aging.

Molecular Biology of the WWOX Gene That Spans Chromosomal Fragile Site FRA16D.

It is now more than 20 years since the FRA16D common chromosomal fragile site was characterised and the WWOX gene spanning this site was identified. In this time, much information has been discovered about its contribution to disease; however, the normal biological role of WWOX is not yet clear. Experiments leading to the identification of the WWOX gene are recounted, revealing enigmatic relationships between the fragile site, its gene and the encoded protein. We also highlight research mainly using the genetically tractable model organism Drosophila melanogaster that has shed light on the integral role of WWOX in metabolism. In addition to this role, there are some particularly outstanding questions that remain regarding WWOX, its gene and its chromosomal location. This review, therefore, also aims to highlight two unanswered questions. Firstly, what is the biological relationship between the WWOX gene and the FRA16D common chromosomal fragile site that is located within one of its very large introns? Secondly, what is the actual substrate and product of the WWOX enzyme activity? It is likely that understanding the normal role of WWOX and its relationship to chromosomal fragility are necessary in order to understand how the perturbation of these normal roles results in disease.

Cp1/cathepsin L is required for autolysosomal clearance in Drosophila.

Macroautophagy/autophagy is a highly conserved lysosomal degradative pathway important for maintaining cellular homeostasis. Much of our current knowledge of autophagy is focused on the initiation steps in this process. Recently, an understanding of later steps, particularly lysosomal fusion leading to autolysosome formation and the subsequent role of lysosomal enzymes in degradation and recycling, is becoming evident. Autophagy can function in both cell survival and cell death, however, the mechanisms that distinguish adaptive/survival autophagy from autophagy-dependent cell death remain to be established. Here, using proteomic analysis of Drosophila larval midguts during degradation, we identify a group of proteins with peptidase activity, suggesting a role in autophagy-dependent cell death. We show that Cp1/cathepsin L-deficient larval midgut cells accumulate aberrant autophagic vesicles due to a block in autophagic flux, yet later stages of midgut degradation are not compromised. The accumulation of large aberrant autolysosomes in the absence of Cp1 appears to be the consequence of decreased degradative capacity as they contain undigested cytoplasmic material, rather than a defect in autophagosome-lysosome fusion. Finally, we find that other cathepsins may also contribute to proper autolysosomal degradation in Drosophila larval midgut cells. Our findings provide evidence that cathepsins play an essential role in the autolysosome to maintain basal autophagy flux by balancing autophagosome production and turnover.Abbreviations: 26-29-p: 26-29kD-proteinase; ADCD: autophagy-dependent cell death; Atg8a: Autophagy-related protein 8a; Cp1/cathepsin L: Cysteine proteinase-1; CtsB: Cathepsin B; cathD: cathepsin D; CtsF: Cathepsin F; GFP: green fluorescent protein; LAMP1: lysosomal-associated membrane protein 1; Mitf: microphthalmia associated transcription factor; PCA: principal component analysis; RNAi: RNA interference; RPF: relative to puparium formation.

Crosstalk between Dpp and Tor signaling coordinates autophagy-dependent midgut degradation.

The majority of developmentally programmed cell death (PCD) is mediated by caspase-dependent apoptosis; however, additional modalities, including autophagy-dependent cell death, have important spatiotemporally restricted functions. Autophagy involves the engulfment of cytoplasmic components in a double membrane vesicle for delivery to the lysosome. An established model for autophagy-dependent PCD is Drosophila larval midgut removal during metamorphosis. Our previous work demonstrated that growth arrest is required to initiate autophagy-dependent midgut degradation and Target of rapamycin (Tor) limits autophagy induction. In further studies, we uncovered a role for Decapentaplegic (Dpp) in coordinating midgut degradation. Here, we provide new data to show that Dpp interacts with Tor during midgut degradation. Inhibiting Tor rescued the block in midgut degradation due to Dpp signaling. We propose that Dpp is upstream of Tor and down-regulation promotes growth arrest and autophagy-dependent midgut degradation. These findings underscore a relationship between Dpp and Tor signaling in the regulation of cell growth and tissue removal.

Dpp regulates autophagy-dependent midgut removal and signals to block ecdysone production.

Animal development and homeostasis require the programmed removal of cells. Autophagy-dependent cell deletion is a unique form of cell death often involved in bulk degradation of tissues. In Drosophila the steroid hormone ecdysone controls developmental transitions and triggers the autophagy-dependent removal of the obsolete larval midgut. The production of ecdysone is exquisitely coordinated with signals from numerous organ systems to mediate the correct timing of such developmental programs. Here we report an unexpected role for the Drosophila bone morphogenetic protein/transforming growth factor ß ligand, Decapentaplegic (Dpp), in the regulation of ecdysone-mediated midgut degradation. We show that blocking Dpp signaling induces premature autophagy, rapid cell death, and midgut degradation, whereas sustained Dpp signaling inhibits autophagy induction. Furthermore, Dpp signaling in the midgut prevents the expression of ecdysone responsive genes and impairs ecdysone production in the prothoracic gland. We propose that Dpp has dual roles: one within the midgut to prevent improper tissue degradation, and one in interorgan communication to coordinate ecdysone biosynthesis and developmental timing.

FOXP3 and miR-155 cooperate to control the invasive potential of human breast cancer cells by down regulating ZEB2 independently of ZEB1.

Control of oncogenes, including ZEB1 and ZEB2, is a major checkpoint for preventing cancer, and loss of this control contributes to many cancers, including breast cancer. Thus tumour suppressors, such as FOXP3, which is mutated or lost in many cancer tissues, play an important role in maintaining normal tissue homeostasis. Here we show for the first time that ZEB2 is selectively down regulated by FOXP3 and also by the FOXP3 induced microRNA, miR-155. Interestingly, neither FOXP3 nor miR-155 directly altered the expression of ZEB1. In breast cancer cells repression of ZEB2, independently of ZEB1, resulted in reduced expression of a mesenchymal marker, Vimentin and reduced invasion. However, there was no de-repression of E-cadherin and migration was enhanced. Small interfering RNAs targeting ZEB2 suggest that this was a direct effect of ZEB2 and not FOXP3/miR-155. In normal human mammary epithelial cells, depletion of endogenous FOXP3 resulted in de-repression of ZEB2, accompanied by upregulated expression of vimentin, increased E-cadherin expression and cell morphological changes. We suggest that FOXP3 may help maintain normal breast epithelial characteristics through regulation of ZEB2, and loss of FOXP3 in breast cancer cells results in deregulation of ZEB2.

WWOX, the chromosomal fragile site FRA16D spanning gene: its role in metabolism and contribution to cancer.

The WWOX gene spans the common chromosomal fragile site FRA16D that is located within a massive (780 kb) intron. The WWOX gene is very long, at 1.1 Mb, which may contribute to the very low abundance of the full-length 1.4 kb mRNA. Alternative splicing also accounts for a variety of aberrant transcripts, most of which are devoid of C-terminal sequences required for WWOX to act as an oxidoreductase. The mouse WWOX gene also spans a chromosomal fragile site implying some sort of functional relationship that confers a selective advantage. The encoded protein domains of WWOX are conserved through evolution (between humans and Drosophila melanogaster) and include WW domains, an NAD -binding site, short-chain dehydrogenase/reductase enzyme and nuclear compartmentalization signals. This homology has enabled functional analyses in D. melanogaster that demonstrate roles for WWOX in reactive oxygen species regulation and metabolism. Indeed the human WWOX gene is also responsive to altered metabolism. Cancer cells typically exhibit altered metabolism (Warburg effect). Many cancers exhibit FRA16D DNA instability that results in aberrant WWOX expression and is associated with poor prognosis for these cancers. It is therefore thought that aberrant WWOX expression contributes to the altered metabolism in cancer. In addition, others have found that a specific (low-expression) allele of WWOX genotype contributes to cancer predisposition.

Common chromosomal fragile site FRA16D tumor suppressor WWOX gene expression and metabolic reprograming in cells.

The WWOX gene spans the FRA16D common chromosomal fragile site and is able to suppress tumor growth. FRA16D is a frequent site of DNA instability in cancer resulting in reduced levels of WWOX expression. Altered levels of WWOX have been shown to affect metabolism. Whereas metabolic reprograming of cells from oxidative phosphorylation to aerobic glycolysis is a major hallmark of tumors, the relationship between common chromosomal fragile site genes and altered metabolism has been unclear. Here we report that altering metabolism from glycolysis to oxidative phosphorylation causes stable increase in steady-state levels of transcripts of the WWOX gene. Consistent with this, exposure to hypoxic conditions, in which cells rely on glycolysis, causes a downregulation of WWOX mRNA. The function of WWOX is therefore intimately integrated with metabolism, as WWOX not only contributes to the metabolic state of cells, its transcript levels are also linked to intracellular metabolic state.

Drosophila orthologue of WWOX, the chromosomal fragile site FRA16D tumour suppressor gene, functions in aerobic metabolism and regulates reactive oxygen species.

Common chromosomal fragile sites FRA3B and FRA16D are frequent sites of DNA instability in cancer, but their contribution to cancer cell biology is not yet understood. Genes that span these sites (FHIT and WWOX, respectively) are often perturbed (either increased or decreased) in cancer cells and both are able to suppress tumour growth. While WWOX has some tumour suppressor characteristics, its normal role and functional contribution to cancer has not been fully determined. We find that a significant proportion of Drosophila Wwox interactors identified by proteomics and microarray analyses have roles in aerobic metabolism. Functional relationships between Wwox and either CG6439/isocitrate dehydrogenase (Idh) or Cu-Zn superoxide dismutase (Sod) were confirmed by genetic interactions. In addition, altered levels of Wwox resulted in altered levels of endogenous reactive oxygen species. Wwox (like FHIT) contributes to pathways involving aerobic metabolism and oxidative stress, providing an explanation for the 'non-classical tumour suppressor' behaviour of WWOX. Fragile sites, and the genes that span them, are therefore part of a protective response mechanism to oxidative stress and likely contributors to the differences seen in aerobic glycolysis (Warburg effect) in cancer cells.

Huntingtin-deficient zebrafish exhibit defects in iron utilization and development.

Huntington's disease (HD) is one of nine neurodegenerative disorders caused by expansion of CAG repeats encoding polyglutamine in their respective, otherwise apparently unrelated proteins. Despite these proteins having widespread and overlapping expression patterns in the brain, a specific and unique subset of neurons exhibits particular vulnerability in each disease. It has been hypothesized that perturbation of normal protein function contributes to the specificity of neuronal vulnerability; however, the normal biological functions of many of these proteins including the HD gene product, Huntingtin (Htt), are unclear. To explore the roles of Htt, we have used antisense morpholino oligonucleotides to observe the effects of Htt deficiency in early zebrafish development. Knockdown of Htt expression resulted in a variety of developmental defects. Most notably, Htt-deficient zebrafish had hypochromic blood due to decreased hemoglobin production, despite the presence of iron within blood cells. Furthermore, transferrin receptor 1 transcripts were increased, suggesting cellular iron starvation. Provision of iron to the cytoplasm in a bio-available form restored hemoglobin production in Htt-deficient embryos. Since erythroid cells acquire iron via receptor-mediated endocytosis of transferrin, these results suggest a role for Htt in making endocytosed iron accessible for cellular utilization. Iron is required for oxidative energy production, and defects in iron homeostasis and energy metabolism are features of HD pathogenesis that are most pronounced in the major region of neurodegeneration. It is therefore plausible that perturbation of Htt's normal role in the iron pathway (by polyglutamine tract expansion) contributes to HD pathology, and particularly to its neuronal specificity.

FRA16D common chromosomal fragile site oxido-reductase (FOR/WWOX) protects against the effects of ionizing radiation in Drosophila.

Fragile sites are chromosomal structures that have been proposed to have a determining role in cancer-associated DNA instability. The human WWOX gene spans the FRA16D chromosomal fragile site, the common minimal region of homozygous deletion found in adenocarcinomas and three out of five translocation breakpoints in multiple myeloma. Transcripts from the alternatively spliced WWOX gene encode proteins with common N-terminal WW domains and variable homology to the oxidoreductase family of proteins. In this study, the Drosophila orthologue of the WWOX gene was identified and subjected to mutagenesis via homologous recombination. The resultant DmWWOX1 mutants were viable but exhibited an increased sensitivity to ionizing radiation. This radiation sensitivity was rescued by reintroduction and expression of either the wild-type Drosophila or human WWOX genes. Thus, the protective function of DmWWOX in response to irradiation in Drosophila is conserved with human WWOX (hWWOX). This is consistent with a protective role for hWWOX where aberrant expression, as a result of breakage at the associated fragile site, could contribute directly to cancer progression.

Common chromosomal fragile site FRA16D mutation in cancer cells.

Neither the molecular basis for common fragile site DNA instability nor the contribution of this form of chromosomal instability to cancer is clearly understood. Fragile site FRA16D (16q23.2) is within regions of frequent loss-of-heterozygosity (LOH) in breast and prostate cancers, is associated with homozygous deletions in various adenocarcinomas and t(14;16) chromosomal translocations in multiple myeloma. The FOR (WWOX) gene spans FRA16D and encodes a partner of p53 that also has a role in apoptosis. Previously untested 53 cancer cell lines were screened for deletions within the FOR/WWOX gene. Deletions were detected in Co115, KM12C and KM12SM. Homozygous deletions in these and two previously identified tumour cell lines were intragenic on both alleles, indicating a distinct mutation mechanism from that causing LOH. Identical FRA16D deletions in two cell lines (one derived from the primary carcinoma and the other from a secondary metastasis) demonstrate that FRA16D DNA instability can be an early, transient event. Sequence analysis across one deletion locates one endpoint within a polymorphic AT-dinucleotide repeat and the other adjacent to an AT-rich mini-satellite repeat implicating AT-rich repeats in FRA16D DNA instability. Another deletion is associated with de novo repetition of the 9 bp AT-rich sequence at one of the deletion endpoints. FRA16D deleted cells retain cytogenetic fragile site expression indicating that the deletions are susceptible sites for breakage rather than regions that confer fragility. Most cell lines with FRA16D homozygous deletions also have FRA3B deletions, therefore common fragile sites represent highly susceptible genome-wide targets for a distinct form of mutation.