Autophagy cooperates with PDGFRA to support oncogenic growth signaling.

Macroautophagy (referred to as autophagy hereafter) is a highly conserved catabolic process which sequesters intracellular substrates for lysosomal degradation. Autophagy-related proteins have been shown to be involved in various aspects of tumor development by engaging with multiple cellular substrates. We recently uncovered a novel role for autophagy in regulating the signaling and levels of PDGFRA, a receptor tyrosine kinase amplified in several cancers. We discovered that PDGFRA can be targeted to autophagic degradation by binding the autophagy cargo receptor SQSTM1. Surprisingly, PDGFRA-mediated signaling is perturbed in the absence of autophagy despite enhanced receptor levels. We show that this is due to disrupted trafficking of the receptor to late endosomes where signaling activity persists. Conversely, prolonged autophagy inhibition results in a transcriptional downregulation of Pdgfra as a result of inhibited signaling activity demonstrating that short- and long-term autophagy inhibition have opposing effects on receptor levels. We further investigated the consequence of PDGFRA regulation by autophagy using a mouse model for gliomagenesis where we observed a disruption in PDGFA-driven tumor formation when autophagy is inhibited. Activation of downstream signaling through Pten mutation overrides the need for autophagy during tumor development suggesting a genotype-specific role for autophagy during tumorigenesis. Altogether, our findings provide a novel mechanism through which autophagy can support tumor growth.

Autophagy supports PDGFRA-dependent brain tumor development by enhancing oncogenic signaling.

Autophagy is a conserved cellular degradation process. While autophagy-related proteins were shown to influence the signaling and trafficking of some receptor tyrosine kinases, the relevance of this during cancer development is unclear. Here, we identify a role for autophagy in regulating platelet-derived growth factor receptor alpha (PDGFRA) signaling and levels. We find that PDGFRA can be targeted for autophagic degradation through the activity of the autophagy cargo receptor p62. As a result, short-term autophagy inhibition leads to elevated levels of PDGFRA but an unexpected defect in PDGFA-mediated signaling due to perturbed receptor trafficking. Defective PDGFRA signaling led to its reduced levels during prolonged autophagy inhibition, suggesting a mechanism of adaptation. Importantly, PDGFA-driven gliomagenesis in mice was disrupted when autophagy was inhibited in a manner dependent on Pten status, thus highlighting a genotype-specific role for autophagy during tumorigenesis. In summary, our data provide a mechanism by which cells require autophagy to drive tumor formation.

Autophagy and autophagy-related pathways in cancer.

Maintenance of protein homeostasis and organelle integrity and function is critical for cellular homeostasis and cell viability. Autophagy is the principal mechanism that mediates the delivery of various cellular cargoes to lysosomes for degradation and recycling. A myriad of studies demonstrate important protective roles for autophagy against disease. However, in cancer, seemingly opposing roles of autophagy are observed in the prevention of early tumour development versus the maintenance and metabolic adaptation of established and metastasizing tumours. Recent studies have addressed not only the tumour cell intrinsic functions of autophagy, but also the roles of autophagy in the tumour microenvironment and associated immune cells. In addition, various autophagy-related pathways have been described, which are distinct from classical autophagy, that utilize parts of the autophagic machinery and can potentially contribute to malignant disease. Growing evidence on how autophagy and related processes affect cancer development and progression has helped guide efforts to design anticancer treatments based on inhibition or promotion of autophagy. In this Review, we discuss and dissect these different functions of autophagy and autophagy-related processes during tumour development, maintenance and progression. We outline recent findings regarding the role of these processes in both the tumour cells and the tumour microenvironment and describe advances in therapy aimed at autophagy processes in cancer.

MYC sensitises cells to apoptosis by driving energetic demand.

The MYC oncogene is a potent driver of growth and proliferation but also sensitises cells to apoptosis, which limits its oncogenic potential. MYC induces several biosynthetic programmes and primary cells overexpressing MYC are highly sensitive to glutamine withdrawal suggesting that MYC-induced sensitisation to apoptosis may be due to imbalance of metabolic/energetic supply and demand. Here we show that MYC elevates global transcription and translation, even in the absence of glutamine, revealing metabolic demand without corresponding supply. Glutamine withdrawal from MRC-5 fibroblasts depletes key tricarboxylic acid (TCA) cycle metabolites and, in combination with MYC activation, leads to AMP accumulation and nucleotide catabolism indicative of energetic stress. Further analyses reveal that glutamine supports viability through TCA cycle energetics rather than asparagine biosynthesis and that TCA cycle inhibition confers tumour suppression on MYC-driven lymphoma in vivo. In summary, glutamine supports the viability of MYC-overexpressing cells through an energetic rather than a biosynthetic mechanism.

LRIG1 is a gatekeeper to exit from quiescence in adult neural stem cells.

Adult neural stem cells (NSCs) must tightly regulate quiescence and proliferation. Single-cell analysis has suggested a continuum of cell states as NSCs exit quiescence. Here we capture and characterize in vitro primed quiescent NSCs and identify LRIG1 as an important regulator. We show that BMP-4 signaling induces a dormant non-cycling quiescent state (d-qNSCs), whereas combined BMP-4/FGF-2 signaling induces a distinct primed quiescent state poised for cell cycle re-entry. Primed quiescent NSCs (p-qNSCs) are defined by high levels of LRIG1 and CD9, as well as an interferon response signature, and can efficiently engraft into the adult subventricular zone (SVZ) niche. Genetic disruption of Lrig1 in vivo within the SVZ NSCs leads an enhanced proliferation. Mechanistically, LRIG1 primes quiescent NSCs for cell cycle re-entry and EGFR responsiveness by enabling EGFR protein levels to increase but limiting signaling activation. LRIG1 is therefore an important functional regulator of NSC exit from quiescence.

Autophagy Brings an END to Aberrant Endocytosis.

Wilfling et al. (2020) characterize a selective autophagy pathway in yeast for early clathrin-mediated endocytosis (CME) proteins facilitated by the phase separation of the CME protein, Ede1, which acts as an intrinsic autophagy receptor for the degradation of Ede1-dependent endocytic protein deposits (ENDs).

The multifaceted functions of ATG16L1 in autophagy and related processes.

Autophagy requires the formation of membrane vesicles, known as autophagosomes, that engulf cellular cargoes and subsequently recruit lysosomal hydrolases for the degradation of their contents. A number of autophagy-related proteins act to mediate the de novo biogenesis of autophagosomes and vesicular trafficking events that are required for autophagy. Of these proteins, ATG16L1 is a key player that has important functions at various stages of autophagy. Numerous recent studies have begun to unravel novel activities of ATG16L1, including interactions with proteins and lipids, and how these mediate its role during autophagy and autophagy-related processes. Various domains have been identified within ATG16L1 that mediate its functions in recognising single and double membranes and activating subsequent autophagy-related enzymatic activities required for the recruitment of lysosomes. These recent findings, as well as the historical discovery of ATG16L1, pathological relevance, unresolved questions and contradictory observations, will be discussed here.

The impact of autophagy during the development and survival of glioblastoma.

Glioblastoma is the most common and aggressive adult brain tumour, with poor median survival and limited treatment options. Following surgical resection and chemotherapy, recurrence of the disease is inevitable. Genomic studies have identified key drivers of glioblastoma development, including amplifications of receptor tyrosine kinases, which drive tumour growth. To improve treatment, it is crucial to understand survival response processes in glioblastoma that fuel cell proliferation and promote resistance to treatment. One such process is autophagy, a catabolic pathway that delivers cellular components sequestered into vesicles for lysosomal degradation. Autophagy plays an important role in maintaining cellular homeostasis and is upregulated during stress conditions, such as limited nutrient and oxygen availability, and in response to anti-cancer therapy. Autophagy can also regulate pro-growth signalling and metabolic rewiring of cancer cells in order to support tumour growth. In this review, we will discuss our current understanding of how autophagy is implicated in glioblastoma development and survival. When appropriate, we will refer to findings derived from the role of autophagy in other cancer models and predict the outcome of manipulating autophagy during glioblastoma treatment.

Membrane targeting of core autophagy players during autophagosome biogenesis.

Autophagosomes are vital organelles required to facilitate the lysosomal degradation of cytoplasmic cargo, thereby playing an important role in maintaining cellular homeostasis. A number of autophagy-related (ATG) protein complexes are recruited to the site of autophagosome biogenesis where they act to facilitate membrane growth and maturation. Regulated recruitment of ATG complexes to autophagosomal membranes is essential for their autophagic activities and is required to ensure the efficient engulfment of cargo destined for lysosomal degradation. In this review, we discuss our current understanding of the spatiotemporal hierarchy between ATG proteins, examining the mechanisms underlying their recruitment to membranes. A particular focus is placed on the relevance of phosphatidylinositol 3-phosphate and the extent to which the core autophagy players are reliant on this lipid for their localisation to autophagic membranes. In addition, open questions and potential future research directions regarding the membrane recruitment and displacement of ATG proteins are discussed here.

Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy.

Autophagy is an essential cellular quality control process that has emerged as a critical one for vascular homeostasis. Here, we show that trichoplein (TCHP) links autophagy with endothelial cell (EC) function. TCHP localizes to centriolar satellites, where it binds and stabilizes PCM1. Loss of TCHP leads to delocalization and proteasome-dependent degradation of PCM1, further resulting in degradation of PCM1's binding partner GABARAP. Autophagic flux under basal conditions is impaired in THCP-depleted ECs, and SQSTM1/p62 (p62) accumulates. We further show that TCHP promotes autophagosome maturation and efficient clearance of p62 within lysosomes, without affecting their degradative capacity. Reduced TCHP and high p62 levels are detected in primary ECs from patients with coronary artery disease. This phenotype correlates with impaired EC function and can be ameliorated by NF-κB inhibition. Moreover, Tchp knock-out mice accumulate of p62 in the heart and cardiac vessels correlating with reduced cardiac vascularization. Taken together, our data reveal that TCHP regulates endothelial cell function via an autophagy-mediated mechanism.

Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling.

Despite recently uncovered connections between autophagy and the endocytic pathway, the role of autophagy in regulating endosomal function remains incompletely understood. Here, we find that the ablation of autophagy-essential players disrupts EGF-induced endocytic trafficking of EGFR. Cells lacking ATG7 or ATG16L1 exhibit increased levels of phosphatidylinositol-3-phosphate (PI(3)P), a key determinant of early endosome maturation. Increased PI(3)P levels are associated with an accumulation of EEA1-positive endosomes where EGFR trafficking is stalled. Aberrant early endosomes are recognised by the autophagy machinery in a TBK1- and Gal8-dependent manner and are delivered to LAMP2-positive lysosomes. Preventing this homeostatic regulation of early endosomes by autophagy reduces EGFR recycling to the plasma membrane and compromises downstream signalling and cell survival. Our findings uncover a novel role for the autophagy machinery in maintaining early endosome function and growth factor sensing.

Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy.

Membrane targeting of autophagy-related complexes is an important step that regulates their activities and prevents their aberrant engagement on non-autophagic membranes. ATG16L1 is a core autophagy protein implicated at distinct phases of autophagosome biogenesis. In this study, we dissected the recruitment of ATG16L1 to the pre-autophagosomal structure (PAS) and showed that it requires sequences within its coiled-coil domain (CCD) dispensable for homodimerisation. Structural and mutational analyses identified conserved residues within the CCD of ATG16L1 that mediate direct binding to phosphoinositides, including phosphatidylinositol 3-phosphate (PI3P). Mutating putative lipid binding residues abrogated the localisation of ATG16L1 to the PAS and inhibited LC3 lipidation. On the other hand, enhancing lipid binding of ATG16L1 by mutating negatively charged residues adjacent to the lipid binding motif also resulted in autophagy inhibition, suggesting that regulated recruitment of ATG16L1 to the PAS is required for its autophagic activity. Overall, our findings indicate that ATG16L1 harbours an intrinsic ability to bind lipids that plays an essential role during LC3 lipidation and autophagosome maturation.

The WD40 domain of ATG16L1 is required for its non-canonical role in lipidation of LC3 at single membranes.

A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double-membrane phagophore, which is driven by the ATG16L1/ATG5-ATG12 complex. In contrast, non-canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3-associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non-canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat-containing C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non-canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD Further, we demonstrate activation of non-canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non-canonical use of autophagy machinery.

Interplay of autophagy, receptor tyrosine kinase signalling and endocytic trafficking.

Vesicular trafficking events play key roles in the compartmentalization and proper sorting of cellular components. These events have crucial roles in sensing external signals, regulating protein activities and stimulating cell growth or death decisions. Although mutations in vesicle trafficking players are not direct drivers of cellular transformation, their activities are important in facilitating oncogenic pathways. One such pathway is the sensing of external stimuli and signalling through receptor tyrosine kinases (RTKs). The regulation of RTK activity by the endocytic pathway has been extensively studied. Compelling recent studies have begun to highlight the association between autophagy and RTK signalling. The influence of this interplay on cellular status and its relevance in disease settings will be discussed here.

PLAA Mutations Cause a Lethal Infantile Epileptic Encephalopathy by Disrupting Ubiquitin-Mediated Endolysosomal Degradation of Synaptic Proteins.

During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.

Suppression of autophagy impedes glioblastoma development and induces senescence.

The function of macroautophagy/autophagy during tumor initiation or in established tumors can be highly distinct and context-dependent. To investigate the role of autophagy in gliomagenesis, we utilized a KRAS-driven glioblastoma mouse model in which autophagy is specifically disrupted via RNAi against Atg7, Atg13 or Ulk1. Inhibition of autophagy strongly reduced glioblastoma development, demonstrating its critical role in promoting tumor formation. Further supporting this finding is the observation that tumors originating from Atg7-shRNA injections escaped the knockdown effect and thereby still underwent functional autophagy. In vitro, autophagy inhibition suppressed the capacity of KRAS-expressing glial cells to form oncogenic colonies or to survive low serum conditions. Molecular analyses revealed that autophagy-inhibited glial cells were unable to maintain active growth signaling under growth-restrictive conditions and were prone to undergo senescence. Overall, these results demonstrate that autophagy is crucial for glioma initiation and growth, and is a promising therapeutic target for glioblastoma treatment.

MicroRNA-26a promotes anoikis in human hepatocellular carcinoma cells by targeting alpha5 integrin.

Metastasis is the major reason for the death of patients suffering from malignant diseases such as human hepatocellular carcinoma (HCC). Among the complex metastatic process, resistance to anoikis is one of the most important steps. Previous studies demonstrate that microRNA-26a (miR-26a) is an important tumor suppressor that inhibits the proliferation and invasion of HCC cells by targeting multiple oncogenic proteins. However, whether miR-26a can also influence anoikis has not been well established. Here, we discovered that miR-26a promotes anoikis of HCC cells both in vitro and in vivo. With a combinational analysis of bioinformatics and public clinical databases, we predicted that alpha5 integrin (ITGA5), an integrin family member, is a putative target of miR-26a. Furthermore, we provide experimental evidence to confirm that ITGA5 is a bona fide target of miR-26a. Through gain- and loss-of-function studies, we demonstrate that ITGA5 is a functional target of miR-26a-induced anoikis in HCC cells. Collectively, our findings reveal that miR-26a is a novel player during anoikis and a potential therapeutic target for the treatment of metastatic HCC.

V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation.

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H(+)-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.

Interaction between FIP200 and ATG16L1 distinguishes ULK1 complex-dependent and -independent autophagy.

Autophagy is a finely orchestrated cellular catabolic process that requires multiple autophagy-related gene products (ATG proteins). The ULK1 complex functions to integrate upstream signals to downstream ATG proteins through an unknown mechanism. Here we have identified an interaction between mammalian FIP200 and ATG16L1, essential components of the ULK1 and ATG5 complexes, respectively. Further analyses show this is a direct interaction mediated by a short domain of ATG16L1 that we term the FIP200-binding domain (FBD). The FBD is not required for ATG16L1 self-dimerization or interaction with ATG5. Notably, an FBD-deleted ATG16L1 mutant is defective in mediating amino acid starvation-induced autophagy, which requires the ULK1 complex. However, this mutant retains its function in supporting glucose deprivation-induced autophagy, a ULK1 complex-independent process. This study therefore identifies a previously uncharacterized interaction between the ULK1 and ATG5 complexes that can distinguish ULK1-dependent and -independent autophagy processes.

Curbing autophagy and histone deacetylases to kill cancer cells.

Cells respond to cytotoxicity by activating a variety of signal transduction pathways. One pathway frequently upregulated during cytotoxic response is macroautophagy (hereafter referred to as autophagy). Previously, we demonstrated that pan-histone deacetylase (HDAC) inhibitors, such as the anticancer agent suberoylanilide hydroxamic acid (SAHA, Vorinostat), can induce autophagy. In this study, we show that HDAC inhibition triggers autophagy by suppressing MTOR and activating the autophagic kinase ULK1. Furthermore, autophagy inhibition can sensitize cells to both apoptotic and nonapoptotic cell death induced by SAHA, suggesting the therapeutic potential of autophagy targeting in combination with SAHA therapy. This study also raised a series of questions: What is the role of HDACs in regulating autophagy? Do individual HDACs have distinct functions in autophagy? How do HDACs regulate the nutrient-sensing kinase MTOR? Since SAHA-induced nonapoptotic cell death is not driven by autophagy, what then is the mechanism underlying the apoptosis-independent death? Tackling these questions should lead to a better understanding of autophagy and HDAC biology and contribute to the development of novel therapeutic strategies.