UVRAG mutations associated with microsatellite unstable colon cancer do not affect autophagy

Reduced levels of autophagy correlate with tumorigenesis, and several inducers of autophagy have been found to be tumor suppressors. One such autophagic inducer is the Beclin 1 binding protein UVRAG, a positive regulator of the class III PI3K/Vps34 complex. UVRAG has been implicated in the formation and maturation of autophagosomes, as well as in endocytic trafficking and suppression of proliferation and in vivo tumorigenicity. In this study we show that approximately one-third of a large series of colon carcinomas with microsatellite instability (MSI ) (n = 102) carry a monoallelic UVRAG mutation, leading to expression of a truncated protein, indicating that this event is involved in tumorigenesis. In order to investigate whether the high incidence of UVRAG mutation in MSI colorectal carcinomas is associated with dysfunctional autophagy we analyzed autophagy levels in several colon cancer cell lines that express wild-type or mutant UVRAG protein. No reduction in autophagy was detected in cell lines expressing mutant UVRAG. Consistent with this, depletion of UVRAG in HE K cells stably expressing GFP-LC3 did not inhibit autophagy, but did decrease epidermal growth factor receptor (EGFR) degradation. Overall our results show that there is no correlation between the presence of the monoallelic UVRAG mutation and inhibition of autophagy. Thus, our data indicate that mechanisms other than autophagy contribute to the tumorigenicity of microsatellite unstable colon carcinomas with monoallelic UVRAG mutation.     

UVRAG in autophagy,inflammation, and cancer

ABSTRACT

Macroautophagy/autophagy deregulation has been observed in perpetuated inflammation and the proliferation of tumor cells. However, the mechanisms underlying these changes have yet to be well-identified. UVRAG is one of the key players of autophagy, but its role in vivo remained puzzling. Our recent study utilized a mouse model with inducible expression of a cancer-derived frameshift (FS) mutation in UVRAG that dominant-negatively inhibits wild-type UVRAG, resulting in impaired stimulus-induced autophagy. The systemically compromised autophagy, particularly mitophagy, notably increases inflammation and associated pathologies. Furthermore, our discovery indicates that time-dependent autophagy suppression and ensuing CTNNB1/β-catenin activation may serve as one tumor-promoting mechanism underpinning age-related cancer susceptibility.     

UVRAG: A New Player in Autophagy and Tumor Cell Growth

Autophagy has a well-documented role in the maintenance of homeostasis and the response to stressful environments and it is often deregulated in various human diseases including cancer. The regulation of the Beclin 1-PI3KC3 complex lipid kinase activity is a critical element in the autophagy signaling pathway. Previous studies1 have demonstrated that Beclin 1-PI3KC3-mediated autophagy is negatively regulated by a proto-oncogene Bcl-2. We have recently identified a novel coiled-coil UVRAG tumor suppressor candidate, which positively engages in Beclin 1-dependent autophagy. UVRAG interacts with Beclin 1, leading to activation of autophagy and thereof inhibition of tumorigenesis. This finding adds a new player to the emerging picture of the autophagy network, underscoring the importance of the coordinated activity between Bcl-2 and UVRAG in the regulation of Beclin 1-PI3KC3- mediated autophagy and tumor cell control.

Addendum to:

Autophagic and Tumor Suppressor Activity of a Novel Beclin 1-Binding Protein UVRAG

Chengyu Liang, Pinghui Feng, Bonsu Ku, Iris Dotan, Dan Canaani, Byung-Ha Oh and Jae U. Jung

Nature Cell Biol 2006; 8:688-99     

UV radiation resistance-associated gene (UVRAG) promotes cell proliferation,migration, invasion by regulating cyclin-dependent kinases (CDK) and integrin-β/Src signaling in breast cancer cells

Breast cancer is a highly heterogeneous group of human cancer with distinct genetic, biological and clinicopathological features. Triple-negative breast cancer (TNBC) is the most aggressive and metastatic type of breast cancer and associated with poor patient survival. However, the role of UV Radiation Resistance-Associated Gene (UVRAG) in TNBC remains unknown. Here, we report that UVRAG is highly upregulated in all TNBC cells and its knockdown leads to the inhibition of cell proliferation, colony formation and progression of cell cycle, which is associated with and reduced expression of cell cycle related protein expression, including Cyclin A2, B1, D1, cdc2 and cdk6 in TNBC cells. Inhibition of UVRAG also suppressed cell motility, migration and invasion of TNBC cells by inhibition of Integrin β1 and β3 and Src activity. Our findings suggest for the first time that UVRAG expression contributes to proliferation, cell cycle progression, motility/migration and invasion of TNBC cells. Thus, targeting UVRAG could be a potential strategy in breast cancer especially against TNBC.

     

A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic

The mammalian class III phosphatidylinositol 3-kinase (PI3K-III) complex regulates fundamental cellular functions, including growth factor receptor degradation, cytokinesis and autophagy. Recent studies suggest the existence of distinct PI3K-III sub-complexes that can potentially confer functional specificity. While a substantial body of work has focused on the roles of individual PI3K-III subunits in autophagy, functional studies on their contribution to endocytic receptor downregulation and cytokinesis are limited. We therefore sought to elucidate the specific nature of the PI3K-III complexes involved in these two processes. High-content microscopy-based assays combined with siRNA-mediated depletion of individual subunits indicated that a specific sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates both receptor degradation and cytokinesis, whereas ATG14L, a PI3K-III subunit involved in autophagy, is not required. The unanticipated role of UVRAG and BIF-1 in cytokinesis was supported by a strong localisation of these proteins to the midbody. Importantly, while the tumour suppressive functions of Beclin 1, UVRAG and BIF-1 have previously been ascribed to their roles in autophagy, these results open the possibility that they may also contribute to tumour suppression via downregulation of mitogenic signalling by growth factor receptors or preclusion of aneuploidy by ensuring faithful completion of cell division.     

Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila

Homotypic fusion and vacuole protein sorting (HOPS) is a tethering complex required for trafficking to the vacuole/lysosome in yeast. Specific interaction of HOPS with certain SNARE (soluble NSF attachment protein receptor) proteins ensures the fusion of appropriate vesicles. HOPS function is less well characterized in metazoans. We show that all six HOPS subunits (Vps11 [vacuolar protein sorting 11]/CG32350, Vps18/Dor, Vps16A, Vps33A/Car, Vps39/CG7146, and Vps41/Lt) are required for fusion of autophagosomes with lysosomes in Drosophila. Loss of these genes results in large-scale accumulation of autophagosomes and blocks autophagic degradation under basal, starvation-induced, and developmental conditions. We find that HOPS colocalizes and interacts with Syntaxin 17 (Syx17), the recently identified autophagosomal SNARE required for fusion in Drosophila and mammals, suggesting their association is critical during tethering and fusion of autophagosomes with lysosomes. HOPS, but not Syx17, is also required for endocytic down-regulation of Notch and Boss in developing eyes and for proper trafficking to lysosomes and eye pigment granules. We also show that the formation of autophagosomes and their fusion with lysosomes is largely unaffected in null mutants of Vps38/UVRAG (UV radiation resistance associated), a suggested binding partner of HOPS in mammals, while endocytic breakdown and lysosome biogenesis is perturbed. Our results establish the role of HOPS and its likely mechanism of action during autophagy in metazoans.     

Essential roles of fibronectin in the development of the left-right embryonic body plan

Studies in Xenopus laevis suggested that cell-extracellular matrix (ECM) interactions regulate the development of the left–right axis of asymmetry; however, the identities of ECM components and their receptors important for this process have remained unknown. We discovered that FN is required for the establishment of the asymmetric gene expression pattern in early mouse embryos by regulating morphogenesis of the node, while cellular fates of the nodal cells, canonical Wnt and Shh signaling within the node were not perturbed by the absence of FN. FN is also required for the expression of Lefty 1/2 and activation of SMADs 2 and 3 at the floor plate, while cell fate specification of the notochord and the floor plate, as well as signaling within and between these two embryonic organizing centers remained intact in FN-null mutants. Furthermore, our experiments indicate that a major cell surface receptor for FN, integrin α5β1, is also required for the development of the left–right asymmetry, and that this requirement is evolutionarily conserved in fish and mice. Taken together, our studies demonstrate the requisite role for a structural ECM protein and its integrin receptor in the development of the left–right axis of asymmetry in vertebrates.     

Beyond autophagy: The role of UVRAG in membrane trafficking

Autophagy is a lysosome-directed membrane trafficking event for the degradation of cytoplasmic components, including organelles. The past few years have seen a great advance in our understanding of the cellular machinery of autophagosome biogenesis, the hallmark of autophagy. However, our global understanding of autophagosome maturity remains relatively poor and fragmented. The topological similarity of autophagosome and endosome delivery to lysosomes suggests that autophagic and endosomal maturation may have evolved to share associated machinery to promote the lysosomal delivery of their cargoes. We have recently discovered that UVRAG, originally identified as a Beclin 1-binding autophagy protein, appears to be an important factor in autophagic and endosomal trafficking through its interaction with the class C Vps tethering complex. Given the ability of UVRAG to bind Beclin 1 and the class C Vps complex in a genetically and functionally separable manner, it may serve as an important regulator for the spatial and/or temporal control of diverse cellular trafficking events. As more non-autophagic functions of UVRAG are unveiled, our understanding of seemingly different cellular processes may move a step further.

Addendum to: Liang C, Lee JS, Inn KS, Gack MU, Li Q, Roberts EA, Vergne I, Deretic V, Feng P, Akazawa C, Jung JU. Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking. Nat Cell Biol 2008; 10:776–87.     

Repression of Rx gene on the left side of the sensory vesicle by Nodal signaling is crucial for right-sided formation of the ocellus photoreceptor in the development of Ciona intestinalis

Nodal signaling plays an essential role in the establishment of left–right asymmetry in various animals. However, it is largely unknown how Nodal signaling is involved in the establishment of the left–right asymmetric morphology. In this study, the role of Nodal signaling in the left–right asymmetric ocellus formation in the ascidian, Ciona intestinalis was dealt with. During the development of C. intestinalis, the ocellus pigment cell forms on the midline and moves to the right side of the midline. Then, the photoreceptor cells form on the right side of the sensory vesicle (SV). Ci-Nodal is expressed on the left side of the SV in the developing tail bud embryo. When Nodal signaling is inhibited, the ocellus pigment cell form but remain on the midline, and expression of marker genes of the ocellus photoreceptor cells is ectopically detected on the left side as well as on the right side of the SV in the larva. Furthermore, Ci-Rx, which is essential for the ocellus differentiation, turns out to be negatively regulated by the Nodal signaling on the left side of the SV, even though it is required for the right-sided photoreceptor formation. These results indicate that Nodal signaling controls the left–right asymmetric ocellus formation in the development of C. intestinalis.     

Subnuclear development of the zebrafish habenular nuclei requires ER translocon function

The dorsal habenular nuclei (Dh) of the zebrafish are characterized by significant left–right differences in gene expression, anatomy, and connectivity. Notably, the lateral subnucleus of the Dh (LsDh) is larger on the left side of the brain than on the right, while the medial subnucleus (MsDh) is larger on the right compared to the left. A screen for mutations that affect habenular laterality led to the identification of the sec61a-like 1(sec61al1) gene. In sec61al1^c163 mutants, more neurons in the LsDh and fewer in the MsDh develop on both sides of the brain. Generation of neurons in the LsDh occurs more rapidly and continues for a longer time period in mutants than in WT. Expression of Nodal pathway genes on the left side of the embryos is unaffected in mutants, as is the left sided placement of the parapineal organ, which promotes neurogenesis in the LsDh of WT embryos. Ultrastructural analysis of the epithalamus indicates that ventricular precursor cells, which form an epithelium in WT embryos, lose apical-basal polarity in sec61al1^c163 mutants. Our results show that in the absence of sec61al1, an excess of precursor cells for the LsDh exit the ventricular region and differentiate, resulting in formation of bilaterally symmetric habenular nuclei.     

Numb regulates the balance between Notch recycling and late-endosome targeting in Drosophila neural progenitor cells

The Notch signaling pathway plays essential roles in both animal development and human disease. Regulation of Notch receptor levels in membrane compartments has been shown to affect signaling in a variety of contexts. Here we used steady-state and pulse-labeling techniques to follow Notch receptors in sensory organ precursor cells in Drosophila. We find that the endosomal adaptor protein Numb regulates levels of Notch receptor trafficking to Rab7-labeled late endosomes but not early endosomes. Using an assay we developed that labels different pools of Notch receptors as they move through the endocytic system, we show that Numb specifically suppresses a recycled Notch receptor subpopulation and that excess Notch signaling in numb mutants requires the recycling endosome GTPase Rab11 activity. Our data therefore suggest that Numb controls the balance between Notch receptor recycling and receptor targeting to late endosomes to regulate signaling output after asymmetric cell division in Drosophila neural progenitors.     

Age-associated and tissue-specific decline in autophagic activity in the nematode C. elegans

Macroautophagy/autophagy is a cellular recycling process that is required for the extended life span observed in many longevity paradigms, including in the nematode C. elegans. However, little is known regarding the spatiotemporal changes in autophagic activity in such long-lived mutants as well as in wild-type animals during normal aging. In a recent study, we report that autophagic activity decreases with age in several major tissues of wild-type C. elegans, including the intestine, body-wall muscle, pharynx, and nerve-ring neurons. Moreover, long-lived daf-2/insulin-signaling mutants and glp-1/Notch receptor mutants display increased autophagic activity, yet with different time- and tissue-specific differences. Notably, the intestine appears to be a critical tissue in which autophagy contributes to longevity in glp-1, but not in daf-2 mutants. Our findings indicate that autophagic degradation is reduced with age, possibly with distinct kinetics in different tissues, and that long-lived mutants increase autophagy in a tissue-specific manner, resulting in increased life span.     

Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking

Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome-endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG-Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy - autophagosome formation and maturation - but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.     

Ral GTPase and the exocyst regulate autophagy in a tissue‐specific manner

Autophagy traffics cellular components to the lysosome for degradation. Ral GTPase and the exocyst have been implicated in the regulation of stress‐induced autophagy, but it is unclear whether they are global regulators of this process. Here, we investigate Ral function in different cellular contexts in Drosophila and find that it is required for autophagy during developmentally regulated cell death in salivary glands, but does not affect starvation‐induced autophagy in the fat body. Furthermore, knockdown of exocyst subunits has a similar effect, preventing autophagy in dying cells but not in cells of starved animals. Notch activity is elevated in dying salivary glands, this change in Notch signaling is influenced by Ral, and decreased Notch function influences autophagy. These data indicate that Ral and the exocyst regulate autophagy in a context‐dependent manner, and that in dying salivary glands, Ral mediates autophagy, at least in part, by regulation of Notch.     

Binding Rubicon to cross the Rubicon

Beclin 1 is an antitumor protein, required for mammalian autophagy, but its precise molecular function is poorly understood. Mass spectrometry analysis reveals that two novel proteins, Atg14L and Rubicon, associate with Beclin 1, together with a known Beclin 1-binding protein, UVRAG. The interactions of Atg14L and UVRAG with the Beclin 1-Vps34 (class III PI3-kinase)-Vps15 core complex are mutually exclusive; Rubicon associates with a subpopulation of UVRAG-containing complexes. The Atg14L complex, which positively regulates autophagy at an early step, localizes to the phagophore/isolation membrane, autophagosome and endoplasmic reticulum. In contrast, the Rubicon-UVRAG complex localizes to the late endosome/lysosome and negatively regulates both autophagy at a later step and the endocytic pathway. Thus, the Beclin 1-Vps34-Vps15 complex functions in autophagy and the endocytic pathway, but its function in a given context depends on the identity of its interacting subunits.     

Highlights from this issue

Germline P granules are specialized protein/RNA aggregates that are found exclusively in germ cells in C. elegans. During the early embryonic divisions that generate germ blastomeres, aggregate-prone P granule components PGL-1 and PGL-3 that remain in the cytoplasm destined for somatic daughters are selectively removed by autophagy. Loss-of-function of components of the autophagy pathway, including the VPS-34/BEC-1 complex, causes accumulation of PGL-1 and PGL-3 into aggregates in somatic cells (termed PGL granules). Formation of PGL granules depends on SEPA-1, which is an integral component of these granules. SEPA-1 is preferentially degraded by autophagy and is also required for the autophagic degradation of PGL-1 and PGL-3. SEPA-1 functions as a bridging molecule in mediating degradation of P granule components by directly interacting with PGL-3 and also with the autophagy protein LGG-1/Atg8. The defect in embryonic development in autophagy mutants is suppressed by mutation of sepa-1, suggesting that autophagic degradation of PGL granule components may provide nutrients for embryogenesis and/or also prevent the formation of aggregates that could be toxic for animal development. Our study reveals a specific physiological function of selective autophagic degradation during C. elegans development.     

Autophagy eliminates cytoplasmic β-catenin and NICD to promote the cardiac differentiation of P19CL6 cells

Autophagy plays important roles in adipogenesis and neuron development. However, how autophagy contributes to cardiac development is not well understood. The main aim of our study was to determine the association between autophagy and myocardial differentiation and its roles in this process. Using a well-established in vitro cardiomyocyte differentiation system, P19CL6 cells, we found that autophagy occurred from the early stage of cardiac differentiation. Blocking autophagy by knocking-down of autophagy-related gene Atg7 or Atg5 inhibited the cardiac differentiation of P19CL6 cells. Further investigation demonstrated that LC3 and P62 could form a complex with β-catenin and NICD, respectively, and promoted the degradation of β-catenin and NICD. Enhancing autophagy promoted the formation of complex, whereas blocking autophagy attenuated the degradation of β-catenin and NICD. Taken together, autophagy could facilitate P19CL6 cells to complete the cardiac differentiation process through blocking Wnt and Notch signaling pathways.     

Autophagy in plants

Autophagy is a highly conserved processing mechanism in eukaryotes whereby cytoplasmic components are engulfed in double-membrane vesicles called autophagosomes and are delivered into organelles such as lysosomes (mammal) or vacuoles (yeast/plant) for degradation and recycling of the resulting molecules. Isolation of yeastAUTOPHAGY (ATG) genes has facilitated the identification of correspondingArabidopsis ATG genes based on sequence similarity. Genetic and molecular analyses using knockout and/or knockdown mutants of those genes have unraveled the biological functions of autophagy during plant development, nutrient recycling, and environmental stress responses. Additional roles for autophagy have been suggested in the degradation of oxidized proteins during oxidative stress and the regulation of hypersensitive response (HR)-programmed cell death (PCD) during innate immunity. Our review summarizes knowledge about the structure and function of autophagic pathways andATG components, and the biological roles of autophagy in plants.     

Acinus: A nuclear regulator of autophagy and endocytic trafficking

Fusion with lysosomes is the common last step of endocytic trafficking and autophagy. Accordingly, several proteins are required in both pathways for cargoes to reach their destinations. Among these proteins, Drosophila Acinus stands out, as it exerts opposite effects on these two pathways, and thus establishes a new paradigm. Loss of Acinus function destabilizes early endosomes, thereby promoting the delivery of their cargo to lysosomes. By contrast, the maturation of autophagosomes to autolysosomes is inhibited in acn mutant cells. The increase in autophagy upon Acinus overexpression and its location to the nucleus are consistent with Acinus being a novel regulator of autophagy.Key words: fat body, endosomes, lysosomes, nuclear protein, Notch signaling, EGF ReceptorMuch of the core machinery that is required for the formation and maturation of autophagosomes and endosomes has been identified by genetic screens in yeast. But as both types of organelles are charged with more complex functions in multicellular organisms, it is not surprising to find additional layers of regulation imposed on them. One such regulatory element was revealed by a genetic screen we conducted in Drosophila.The screen''s original idea was to take advantage of the observation that many proteins acting in trafficking to lysosomes also function in the biogenesis of lysosome-related organelles. Among these, the pigment granules—responsible for the characteristic color of the fly eye—are easily scored for defects. Thus, we set up a primary screen for eye color mutants. Among the more than 500 original hits, a secondary screen identified those mutants that altered endocytic trafficking. Importantly, the genetic tool kit assembled by the fly community allowed us to screen homozygous mutant eyes in otherwise heterozygous flies. This schema made it possible to identify mutations that are homozygous lethal as one might expect for null alleles of genes required for lysosomal delivery.One of the unexpected genes identified by this screen was acinus (acn). The Acn protein lacks any domain signatures and is most similar to human Acinus, which had been implicated in the destruction of chromatin during apoptosis. It is not clear yet whether the Drosophila protein contributes to this function as well, but in acn null alleles chromatin condensation and fragmentation during apoptosis appear normal.There is, however, a profound effect on endocytic trafficking, as acn is required for stabilization of early endosomes. Staining for endocytosed ligands, such as Boss or Delta, is drastically reduced, concomitant with a reduction in early endosomes marked by Rab5 or the SNARE Avl. By contrast, late endosomes marked by Rab7 appear normal. These changes do not represent a block in the initial internalization of the ligands, as inhibition of lysosomal degradation reveals the same accumulation of internalized ligands in wild-type and acn mutant cells.Reduced stability of early endosomes also causes reduced signaling from EGF receptors and Notch, consistent with the emerging notion that signaling from these receptors may be linked to their uptake into early endosomes.Many mutants that disrupt endocytic trafficking also affect autophagy. We found that this theme extends to acn. The most accessible form of autophagy in Drosophila is found in fat bodies after a short period of starvation. Activation of the AKT1/TOR pathway triggers the formation of autophagosomes, which mature into autolysosomes by fusing with lysosomes. Loss of acn interferes with this maturation step, as shown by the reduction in LysoTracker staining and also by quantitative electron microscopy. Consistent with an effect on the maturation of autophagosomes, acn is required downstream of TOR signaling. For example, expression of dominant-negative TOR kinase is a powerful tool to induce autophagy in the fat body of wild-type, but not acn larvae.Interestingly, overexpression of Acn induces autophagy. This does not appear to be merely a side effect. Ubiquitous expression of Acn is lethal, but flies survive when autophagy is suppressed by knockdown of ATG5, a core element of the autophagy machinery. We find that this enhanced autophagy is also independent of the TOR pathway.Taken together, this analysis of the first null mutant of an acinus gene in any system reveals its function as a regulator of endosomal and autophagosomal dynamics, modulating developmental signaling and the cellular response to starvation. Our investigation of acn loss-of-function phenotypes reveals defects in membrane trafficking during endocytosis and autophagy. We were therefore surprised that Acn protein localized to the nucleus, and that we failed to detect any consistent localization to endocytic or autophagic structures. This unexpected finding was further tested with transgenes expressing Myc-tagged Acn in the context of a genomic rescue construct. This tagged protein, under control of its endogenous enhancer/promoter elements, rescued all aspects of Acn function, and, nevertheless, localized to the nucleus, rather than any endosomal compartment.These findings suggest that the mechanism by which Acinus proteins modify endocytosis and autophagy may be indirect. One model for such an indirect effect is suggested by the interaction of mammalian Acinus proteins with several RNA binding proteins. Modulation of the levels or structure of RNAs that encode specific elements of the endocytosis or autophagy pathways may constitute an exciting new element of their regulation. Testing this possibility and identifying potential targets regulated by this Acn-dependent mechanism are important challenges that we have just begun to address.