Modulation of intracellular ROS levels by TIGAR controls autophagy

The p53‐inducible TIGAR protein functions as a fructose‐2,6‐bisphosphatase, promoting the pentose phosphate pathway and helping to lower intracellular reactive oxygen species (ROS). ROS functions in the regulation of many cellular responses, including autophagy—a response to stress conditions such as nutrient starvation and metabolic stress. In this study, we show that TIGAR can modulate ROS in response to nutrient starvation or metabolic stress, and functions to inhibit autophagy. The ability of TIGAR to limit autophagy correlates strongly with the suppression of ROS, with no clear effects on the mTOR pathway, and is p53 independent. The induction of autophagy in response to loss of TIGAR can function to moderate apoptotic response by restraining ROS levels. These results reveal a complex interplay in the regulation of ROS, autophagy and apoptosis in response to TIGAR expression, and shows that proteins similar to TIGAR that regulate glycolysis can have a profound effect on the autophagic response through ROS regulation.     

Identification of secreted proteins that reflect autophagy dynamics within tumor cells

Macroautophagy, a catabolic process of cellular self-digestion, is an important tumor cell survival mechanism and a potential target in antineoplastic therapies. Recent discoveries have implicated autophagy in the cellular secretory process, but potential roles of autophagy-mediated secretion in modifying the tumor microenvironment are poorly understood. Furthermore, efforts to inhibit autophagy in clinical trials have been hampered by suboptimal methods to quantitatively measure tumor autophagy levels. Here, we leveraged the autophagy-based involvement in cellular secretion to identify shed proteins associated with autophagy levels in melanoma. The secretome of low-autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu in physiological 3-dimensional cell culture using quantitative proteomics. These comparisons identified candidate autophagy biomarkers IL1B (interleukin 1, β), CXCL8 (chemokine (C-X-C motif) ligand 8), LIF (leukemia inhibitory factor), FAM3C (family with sequence similarity 3, member C), and DKK3 (dickkopf WNT signaling pathway inhibitor 3) with known roles in inflammation and tumorigenesis, and these proteins were subsequently shown to be elevated in supernatants of an independent panel of high-autophagy melanoma cell lines. Secretion levels of these proteins increased when low-autophagy melanoma cells were treated with the autophagy-inducing tat-BECN1 (Beclin 1) peptide and decreased when ATG7 (autophagy-related 7) was silenced in high-autophagy cells, thereby supporting a mechanistic link between these secreted proteins and autophagy. In addition, serum from metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from patients with low-autophagy tumors. These results suggest that autophagy-related secretion affects the tumor microenvironment and measurement of autophagy-associated secreted proteins in plasma and possibly in tumors can serve as surrogates for intracellular autophagy dynamics in tumor cells.     

Identification of secreted proteins that reflect autophagy dynamics within tumor cells

Macroautophagy, a catabolic process of cellular self-digestion, is an important tumor cell survival mechanism and a potential target in antineoplastic therapies. Recent discoveries have implicated autophagy in the cellular secretory process, but potential roles of autophagy-mediated secretion in modifying the tumor microenvironment are poorly understood. Furthermore, efforts to inhibit autophagy in clinical trials have been hampered by suboptimal methods to quantitatively measure tumor autophagy levels. Here, we leveraged the autophagy-based involvement in cellular secretion to identify shed proteins associated with autophagy levels in melanoma. The secretome of low-autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu in physiological 3-dimensional cell culture using quantitative proteomics. These comparisons identified candidate autophagy biomarkers IL1B (interleukin 1, β), CXCL8 (chemokine (C-X-C motif) ligand 8), LIF (leukemia inhibitory factor), FAM3C (family with sequence similarity 3, member C), and DKK3 (dickkopf WNT signaling pathway inhibitor 3) with known roles in inflammation and tumorigenesis, and these proteins were subsequently shown to be elevated in supernatants of an independent panel of high-autophagy melanoma cell lines. Secretion levels of these proteins increased when low-autophagy melanoma cells were treated with the autophagy-inducing tat-BECN1 (Beclin 1) peptide and decreased when ATG7 (autophagy-related 7) was silenced in high-autophagy cells, thereby supporting a mechanistic link between these secreted proteins and autophagy. In addition, serum from metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from patients with low-autophagy tumors. These results suggest that autophagy-related secretion affects the tumor microenvironment and measurement of autophagy-associated secreted proteins in plasma and possibly in tumors can serve as surrogates for intracellular autophagy dynamics in tumor cells.     

Autophagy: Mechanisms, regulation, and its role in tumorigenesis

Autophagy (from Greek “auto” — self, “phagos” — to eat) is the major catabolic process involved in the delivery and lysosomal degradation of long-lived intracellular components: proteins, lipids, nucleic acids, and organelles. Since the discovery of genes involved in regulation of autophagy in the 1990s, there has been a significant increase in studies of autophagy as a process involved in maintaining cellular homeostasis, as well as its role in the development of different pathologies. This review focuses on the basics of autophagy and its regulatory mechanisms. The role of autophagy in the maintenance of cellular homeostasis and tumorigenesis is also discussed.     

Species‐specific impact of the autophagy machinery on Chikungunya virus infection

Chikungunya virus (CHIKV) is a recently re‐emerged arbovirus that triggers autophagy. Here, we show that CHIKV interacts with components of the autophagy machinery during its replication cycle, inducing a cytoprotective effect. The autophagy receptor p62 protects cells from death by binding ubiquitinated capsid and targeting it to autophagolysosomes. By contrast, the human autophagy receptor NDP52—but not its mouse orthologue—interacts with the non‐structural protein nsP2, thereby promoting viral replication. These results highlight the distinct roles of p62 and NDP52 in viral infection, and identify NDP52 as a cellular factor that accounts for CHIKV species specificity.     

Autophagy process is associated with anti-neoplastic function

Autophagy is a highly conserved process of cellular degradation, which is present in yeast, plants, and mammals. Under normal physiological conditions, autophagy acts to maintain cellular homeostasis and regulate the turnover of organelles. In response to cellular stresses, autophagy prevents the accumulation of impaired proteins and organelles, which serves to inhibit carcinogenesis. On this basis, it is widely accepted that most tumor suppressors, such as beclin 1 associated proteins, forkhead box class O (FoxO) family proteins, multiple mammalian target of Rapamycin (mTOR) inactivators, and nuclear p53 play a role in inducing autophagy. Here, we focus on how the process of autophagy is associated with anti-neoplastic function.     

Inhibitive effects of Photofrin on cellular autophagy

Depending on the circumstances, autophagy can be either a protective or damaging cellular process. The role of autophagy in photodynamic therapy (PDT), a photo‐chemotherapy that utilizes light to activate a photosensitizer drug to achieve localized cellular damage, has been explored in recent years. It has been reported that autophagy in PDT is significantly influenced by the treatment protocol. In this work, the role of Photofrin, a well‐established clinical photosensitizer, in regulating cellular autophagy was investigated. The effects of Photofrin on cellular autophagy induced by conventional starvation or rapamycin techniques were studied. By fluorescence imaging, Western blotting and cell viability assays, it was found that Photofrin can effectively inhibit cellular autophagy induced by starvation or rapamycin. This autophagy blocking is independent of the photosensitizing property of the drug. With Baf‐A1, a well‐established agent that inhibits autophagosome from fusing with lysosome, we also found that, the observed phenomenon is not due to accelerated degradation of existing autophagosomes, thus proving that the drug Photofrin alone, without light excitation, can truly block autophagy. J. Cell. Physiol. 224: 414–422, 2010. © 2010 Wiley‐Liss, Inc.     

An appetite for destruction

Autophagy plays an important role in cellular survival by resupplying cells with nutrients during starvation or by clearing misfolded proteins and damaged organelles and thereby preventing degenerative diseases. Conversely, the autophagic process is also recognized as a cellular death mechanism. The circumstances that determine whether autophagy has a beneficial or a detrimental role in cellular survival are currently unclear. We recently showed that autophagy induction is detrimental in neurons that lack a functional AMPK enzyme (AMP-activated protein kinase) and that suffer from severe metabolic stress. We further demonstrated that autophagy and AMPK are interconnected in a negative feedback loop that prevents excessive and destructive stimulation of the autophagic process. Finally, we uncovered a new survival mechanism in AMPK-deficient neurons—cell cannibalism.     

受体酪氨酸激酶对自噬的调控及其研究进展*

自噬是真核生物进化上保守的溶酶体降解的生物学过程,在维护细胞内的稳态、消除有害组分等方面起到了重要作用。受体酪氨酸激酶家族(receptor tyrosine kinase,RTKs)是一类激酶蛋白,在正常细胞和癌症细胞的运动和侵袭中起着重要作用。RTKs蛋白既能促进自噬,也能抑制自噬。研究显示,RTKs能够在肿瘤和相关疾病中发挥自噬作用,比如表皮生长因子受体(epidermal growth factor receptor,EGFR)可以抑制自噬,从而促进肿瘤生长、增殖;还能通过RTK/Ras/ERK信号通路诱导自噬,进而参与诸如细胞免疫反应之类的相关疾病。主要综述了RTKs对自噬的调控作用和相关研究成果,为靶点靶向疗法的理论依据提供了基础。     

Autophagy protects auditory hair cells against neomycin-induced damage

Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.     

A role for mitochondria in autophagy regulation

Understanding the functional relationship between mitochondria and autophagy is critical for understanding the molecular mechanisms underlying aging and neurodegeneration. Autophagy functions in both cellular homeostasis and in quality control in the selective removal of dysfunctional mitochondria. A current working model in the field is that impaired autophagy results in a cell-damaging accumulation of dysfunctional mitochondria over time. We described our findings that respiratory-deficient mitochondria can inhibit general (macro) autophagy in Saccharomyces cerevisiae by conserved signaling pathways during amino acid starvation. These data point to an interdependence of mitochondrial function and autophagy and raise the possibility that negative regulation of autophagy by dysfunctional mitochondria is a critical contributing factor in many diseases.     

Oxidized LDLs trigger endoplasmic reticulum stress and autophagy: prevention by HDLs

Oxidized LDLs (oxLDLs) induce various cellular dysfunctions potentially implicated in the pathogenesis of atherosclerosis. For instance, toxic concentrations of oxLDLs trigger ER stress, autophagy and apoptosis. High-density lipoproteins (HDLs) counteract several adverse biological effects triggered by oxLDLs. Our recent study reveals that HDLs inhibit the activation of ER stress and of autophagy induced by oxLDLs.     

A role for mitochondria in autophagy regulation

Understanding the functional relationship between mitochondria and autophagy is critical for understanding the molecular mechanisms underlying aging and neurodegeneration. Autophagy functions in both cellular homeostasis and in quality control in the selective removal of dysfunctional mitochondria. A current working model in the field is that impaired autophagy results in a cell-damaging accumulation of dysfunctional mitochondria over time. We described our findings that respiratory-deficient mitochondria can inhibit general (macro) autophagy in Saccharomyces cerevisiae by conserved signaling pathways during amino acid starvation. These data point to an interdependence of mitochondrial function and autophagy and raise the possibility that negative regulation of autophagy by dysfunctional mitochondria is a critical contributing factor in many diseases.     

Inhibition of autophagy impairs tumor cell invasion in an organotypic model

Autophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. It contributes to energy and organelle homeostasis and the preservation of proteome and genome integrity. Although a role in cancer is unquestionable, there are conflicting reports that autophagy can be both oncogenic and tumor suppressive, perhaps indicating that autophagy has different roles at different stages of tumor development. In this report, we address the role of autophagy in a critical stage of cancer progression—tumor cell invasion. Using a glioma cell line containing an inducible shRNA that targets the essential autophagy gene Atg12, we show that autophagy inhibition does not affect cell viability, proliferation or migration but significantly reduces cellular invasion in a 3D organotypic model. These data indicate that autophagy may play a critical role in the benign to malignant transition that is also central to the initiation of metastasis.     

Gemcitabine Induces Poly (ADP-Ribose) Polymerase-1 (PARP-1) Degradation through Autophagy in Pancreatic Cancer

Poly (ADP-ribose) polymerase-1 (PARP-1) and autophagy play increasingly important roles in DNA damage repair and cell death. Gemcitabine (GEM) remains the first-line chemotherapeutic drug for pancreatic cancer (PC). However, little is known about the relationship between PARP-1 expression and autophagy in response to GEM. Here we demonstrate that GEM induces DNA-damage response and degradation of mono-ADP ribosylated PARP-1 through the autophagy pathway in PC cells, which is rescued by inhibiting autophagy. Hypoxia and serum starvation inhibit autophagic activity due to abrogated GEM-induced mono-ADP-ribosylated PARP-1 degradation. Activation of extracellular regulated protein kinases (ERK) induced by serum starvation shows differences in intracellular localization as well as modulation of autophagy and PARP-1 degradation in GEM-sensitive KLM1 and -resistant KLM1-R cells. Our study has revealed a novel role of autophagy in PARP-1 degradation in response to GEM, and the different impacts of MEK/ERK signaling pathway on autophagy between GEM-sensitive and -resistant PC cells.     

Oxidized LDLs trigger endoplasmic reticulum stress and autophagy: Prevention by HDLs

Oxidized LDLs (oxLDLs) induce various cellular dysfunctions potentially implicated in the pathogenesis of

atherosclerosis. For instance, toxic concentrations of oxLDLs trigger ER stress, autophagy and apoptosis. High-density lipoproteins (HDLs) counteract several adverse biological effects triggered by oxLDLs. Our recent study reveals that HDLs inhibit the activation of ER stress and of autophagy induced by oxLDLs.     

A Ypt/Rab GTPase module makes a PAS

Organization of membrane micro-domains by Ypt/Rab GTPases is key for all membrane trafficking events in eukaryotic cells. Since autophagy is a membrane trafficking process, it was expected that these GTPases would play a role in autophagy as well. While evidence about participation of Ypt/Rabs in autophagy is beginning to emerge, the mechanisms by which they act in this process are still not clear. Moreover, it is still questionable if and how Ypt/Rabs coordinate autophagy with other cellular trafficking processes. Yeast Ypt1 and its mammalian homolog Rab1 are required for both endoplasmic reticulum (ER)-to-Golgi transport and autophagy, suggesting that they coordinate these two processes. In our recent paper, we identify Atg11, a bona fide phagophore assembly site (PAS) component, as a downstream effector of Ypt1. Moreover, we show that three components of a GTPase module—the Ypt1 activator, Trs85-containing TRAPP complex, Ypt1, and the Atg11 effector—interact on the PAS and are required for PAS formation during selective autophagy. We propose that Ypt/Rabs coordinate the secretory and the autophagic pathways by recruiting process-specific effectors.     

Visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus

Autophagy is a highly conserved cellular response to starvation that leads to the degradation of organelles and long-lived proteins in lysosomes and is important for cellular homeostasis, tissue development and as a defense against aggregated proteins, damaged organelles and infectious agents. Although autophagy has been studied in many animal species, reagents to study autophagy in avian systems are lacking. Microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) is an important marker for autophagy and is used to follow autophagosome formation. Here we report the cloning of avian LC3 paralogs A, B and C from the domestic chicken, Gallus gallus domesticus, and the production of replication-deficient, recombinant adenovirus vectors expressing these avian LC3s tagged with EGFP and FLAG-mCherry. An additional recombinant adenovirus expressing EGFP-tagged LC3B containing a G120A mutation was also generated. These vectors can be used as tools to visualize autophagosome formation and fusion with endosomes/lysosomes in avian cells and provide a valuable resource for studying autophagy in avian cells. We have used them to study autophagy during replication of infectious bronchitis virus (IBV). IBV induced autophagic signaling in mammalian Vero cells but not primary avian chick kidney cells or the avian DF1 cell line. Furthermore, induction or inhibition of autophagy did not affect IBV replication, suggesting that classical autophagy may not be important for virus replication. However, expression of IBV nonstructural protein 6 alone did induce autophagic signaling in avian cells, as seen previously in mammalian cells. This may suggest that IBV can inhibit or control autophagy in avian cells, although IBV did not appear to inhibit autophagy induced by starvation or rapamycin treatment.