Synonyms

Autophagocytosis; Cellular self-cannibalism; Cellular self-digestion; Macroautophagy

Definition

Autophagy is the intracellular uptake of cytoplasm (proteins, nucleic acids, small molecules, whole organelles, etc.) into the lysosome and its subsequent degradation. Autophagy is a constitutive as well as a stress-inducible process responsible for the degradation of the majority of cellular proteins.

Characteristics

The lysosomal uptake and degradation of proteins by autophagy can be found in virtually all eukaryotic cells. Autophagy is a homeostatic catabolic process by which long-lived cytosolic proteins and complexes (like ribosomes) are degraded and recycled. Unlike the ubiquitin-proteosome system of degradation, autophagy is able to degrade large protein aggregates and is the only pathway able to degrade whole organelles. Autophagy is regarded to be a largely nonselective bulk process, but it also exhibits selectivity during the biogenesis of the lysosome (import of lysosomal hydrolases), cellular differentiation and cell death, the engulfment of certain bacteria and viruses (xenophagy), and the elimination of mitochondria (mitophagy), peroxisomes (pexophagy), lipid droplets (lipophagy), Golgi complexes, and the endoplasmic reticulum.

Autophagy can be upregulated as a response to nutrient deprivation, growth factor withdrawal, during stress response (oxidative stress, chemotherapy, radiation, protein aggregation), developmental differentiation, and tumor suppression. Autophagy has been shown to have far-reaching health effects including adaptive immunity, anti-inflammatory, microbial infections, heart disease, neurodegeneration, and cancer.

Autophagy shows high conservation throughout evolution, and autophagic degradation is being studied in model organisms like Dictyostelium discoideum, Caenorhabditis elegans, Drosophila, and mice. Most of the autophagy genes (ATG genes) have been discovered in the yeast Saccharomyces cerevisiae. Many of them are conserved in higher eukaryotes including mammals.

Cell Biology of Autophagy

There are three morphologically distinct forms of autophagy (Fig. 1): (i) Chaperone-mediated autophagy involves the recognition of cytosolic proteins by a chaperone-related receptor and its subsequent, direct translocation through the lysosomal membrane. (ii) In microautophagy, cytosolic material is sequestered through invaginations of the lysosome. (iii) In macroautophagy, cytosol is engulfed by a double membrane vesicle, thereby delivering the inner vesicle (autophagic body) of the autophagosomes into the lysosome. In subsequent steps, the vesicle membrane and the autophagic cargo are degraded and amino acids and other small molecules are recycled. Macroautophagy is the most prominent form of autophagy; therefore these terms are often used synonymously.

Fig. 1
figure 1

Cell biology of three forms of autophagy. In macroautophagy (hereafter referred to simply as autophagy), intracellular membranes form in the process of sequestering cytosolic material. The edges of these isolation membranes fuse to form mature double membrane structures (autophagosomes). The outer membranes of the autophagosomes fuse with the lysosome. Delivery of the inner sequestered material leads to the appearance of autophagic bodies within the lysosome. Phospholipases, proteases, and other hydrolases degrade intralysosomal membranes and their content for reuse. Constitutive autophagy aids in maintaining homeostasis by replenishing the cellular storages of energy and building blocks. Microautophagy involves the direct uptake of cytosol through membrane invaginations of the lysosome. In chaperone mediated autophagy, the autophagic cargo containing a specific pentapeptide motif is recognized in the cytosol by the carrier chaperone heat shock cognate 70 (Hsc70). Hsc70 guides the substrate protein to the lysosome where it is translocated through the membrane via the lysosome-associated membrane protein type 2A (LAMP2A) receptor

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Autophagosome formation

To date, more than 30 Atg proteins have been identified and about half of them play an essential role during formation of the autophagosome. The others are specific for autophagy subtypes such as mitophagy and pexophagy. The core machinery can be subdivided into several functional groups. Autophagy is initiated by the ULK1/2 kinase complex which consists of the Ser/Thr kinase ULK1/2, FIP200, Atg13, and Atg101. During starvation, Atg13 and ULK1/2 are dephosphorylated and able to interact. They then form a complex with FIP200 which ULK1/2 phosphorylates, thus inducing formation of the autophagosome. Autophagy initiation also requires phosphatidylinositol-3-phosphate (PI3P). This is produced by the phosphatidylinositol-3 kinase (PI3K) complex I, which is comprised of the class III PI3K hVps34, Beclin1, Atg14L, and p150. Beclin1 can also interact with UVRAG and Bif-1 to stimulate autophagy. WIPI-1, a PI3P effector, forms a complex with Atg9, which is the sole integral membrane protein of the core complex machinery and shuttles between the site of autophagosome formation and cytoplasmic pools. Expansion of the isolation membranes during autophagosome formation requires two ubiquitin-like conjugation systems. In the first system, Atg12 is irreversibly conjugated to Atg5 via Atg7 and Atg10, the E1- and E2-like enzymes, respectively. Atg5 then interacts noncovalently with Atg16L1. Dimerization of Atg16L1 via its coiled coil domain causes dimerization of the entire Atg16L1-Atg5-Atg12 complex. In the second conjugation system, the ubiquitin-like protein LC3 is first processed at its C-terminus by Atg4 and then conjugated to the lipid phosphatidyl ethanolamine (PE) via Atg7 and Atg3, its E1- and E2-like enzymes. Conjugation of LC3 to PE is stimulated by the Atg16L1-Atg5-Atg12 complex which functions as its E3-like enzyme. The Atg16L1-Atg5-Atg12 complex dissociates from the membrane either directly before or fusion of the leading edges of the isolation membrane to form the mature autophagosome. Atg4 cleaves LC3 from the outer membrane of the autophagosome. However, since LC3 still remains attached to the inner membrane of the mature autophagosome it can be used as a marker for tracking mature autophagosomes.

Signal Transduction

When nutrients, amino acids in particular, are abundant, autophagic activity is reduced to basal levels. Concomitantly, active growth factor signaling downregulates autophagy to basal levels. Many times, the transduction of these signals is accomplished through the central regulator of autophagy, the kinase mammalian target of rapamycin complex 1 (mTORC1) (Fig. 2). Extracellular growth factor or hormone (e.g., insulin) signaling occurs through receptor tyrosine kinases which activate the class I PI3K/AKT/PKB kinase pathway and causes the phosphorylation and subsequent inactivation of tuberous sclerosis complex (TSC). TSC then loses its ability to act as the GTPase activating protein (GAP) for Ras homolog enriched in brain (Rheb), a GTPase which has an inhibitory effect on mTORC1. In this way, mTORC1 acts as a nutrient sensor to control cell growth via translational and transcriptional mechanisms and inhibit autophagy under nutrient-rich conditions. Deregulation of this pathway has been linked to various cancers through several molecules. Mutations in PI3K which cause it to be constitutively active are often found in human cancers and cause inhibition of autophagy. PTEN normally inhibits signaling of the class I PI3K to AKT kinase. Haplo-insuffiency of PTEN results in activation of AKT kinase and suppression of autophagy in cancer cells. PTEN haplo-insufficiency together with Rheb overexpression stimulates tumorigenesis in prostate cancer. Furthermore, increased mTORC1 signaling triggers protein cell growth and proliferation, thus enhancing tumorigenesis.

Fig. 2
figure 2

Regulation of autophagy in response to growth factor signaling and nutrient availability. In the presence of growth factors and nutrients, the mTOR kinase complex (mTORC1) is activated through PI3K and AKT and by amino acids. TOR inhibits autophagy and induces translation of household genes. Under these conditions, anabolic processes like translation and cell growth are induced, whereas β-oxidation and autophagic turnover are repressed. The small GTPase Rheb and its GTPase activators, the tuberous sclerosis complex (TSC) proteins are involved upstream in TOR signaling. Autophagy can also be stimulated by the RAS/RAF/MEK/ERK pathway. Cancers with mutations in this pathway are constitutively active and increased autophagy can be used as a means to promote tumor survival.

Low intracellular energy levels are detected by an increase in the AMP to ATP ratio through the LKB1 kinase and the AMP-activated protein kinase (AMPK). AMPK in turn phosphorylates TSC and regulatory associated protein of mTOR (Raptor). Proximate induction of autophagy by formation of the isolation membrane is initiated by the ULK1/2 kinase complex which consists of the Ser/Thr kinase ULK1/2 in complex with FIP200, Atg13, and Atg101. In the absence of growth factor signaling and under conditions of nutrient deprivation, cell survival is ensured by basal levels of autophagy. Cell surface nutrient expression is shut down and TOR kinase is inactive. Autophagy provides energy from within the cell by recycling as an alternative to external sources. Excess autophagy can also be associated with cell death

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mTORC1 also regulates metabolism through the LKB1-AMPK pathway. AMPK is also thought to be involved in tumorigenesis and cancer cell metabolism. Low intracellular energy level and metabolic stress can be detected by an increase in the AMP to ATP ratio. This is detected by the LKB1 kinase which phosphorylates and thereby activates AMP-activated protein kinase (AMPK). AMPK in turn phosphorylates TSC and regulatory associated protein of mTOR (Raptor). This results in the inhibition of mTORC1 and the subsequent stimulation of autophagy as a prosurvival mechanism when faced with metabolic stress. The LKB1-AMPK signaling pathway also causes the phosphorylation and stabilization of the cyclin-dependent kinase inhibitor p27kip, a factor which is required for glucose starvation-induced autophagy as well as energy conservation through the induction of cell cycle arrest.

The transcription factor p53 acts as a tumor suppressor and is mutated in about half of all human cancers. Normally p53 is upregulated in response to DNA damage, oncogenic stress, and hypoxia, all of which are stress conditions commonly occurring in cancer. In mammalian cells, there are two pools of p53 with opposing functions in the regulation of autophagy. Cytoplasmic p53 inhibits autophagy. However, when p53 is translocated into the nucleus it exerts a stimulatory effect on autophagy by targeting genes inhibitory to mTORC1 such as such AMPKβ, TSC2, and PTEN. Nuclear p53 also stimulates autophagy and apoptosis upon genotoxic stress by targeting damage-regulated autophagy modulator (DRAM) gene. The downregulation of DRAM in cancer cells has been observed.

Another regulatory pathway in autophagy that has been implicated in cancer involves members of the antiapoptotic Bcl-2 family: Bcl-2, Bcl-xL, and Mc1. These proteins negatively regulate autophagy by binding to Beclin1, thereby inhibiting its interaction with the PI3K complex which is required for autophagy initiation. Under starvation conditions, c-Jun N-terminal kinase (JNK) phosphorylates Bcl-2 which causes its dissociation from Beclin1. This frees Beclin1 to interact with Vps34 and the PI3K complex to induce autophagy. The tumor suppressor death associated protein kinase (DAPK1) stimulates autophagy when it phosphorylates Beclin1, causing it to dissociate from Bcl-xL. The tumor suppressor ARF binds to Bcl-xL thus freeing Beclin1 to participate in autophagy initiation. Bcl-2 family members are often overexpressed in cancer cells and therefore able to inhibit autophagy. Furthermore, the knockdown of Bcl-2 stimulates autophagy and apoptosis in tumor cells.

Autophagy and Cancer

The complex role of autophagy in cancer has been called a double-edged sword. On the one hand, autophagy functions as a tumor suppressor, but on the other hand, it also serves as a cell survival mechanism, which can also be applied to tumor cells. Which path it ultimately takes depends on the cellular context, the source and degree of stress, and which signaling pathways have been deregulated.

In the initiating stages of of cancer, autophagy is more likely to act in a tumor-suppressive role. Its housekeeping functions of maintaining functional mitochondriaand mitigating oxidative stress help suppress tumor progression. Loss of autophagy can result in accumulation of p62 protein aggregates, reactive oxygen species, DNA damage, inflammation, signaling deregulation, and other hallmarks seen in cancer initiation and progression. Furthermore, several proteins which have been observed as tumor suppressors are either essential to the core complex machinery (Atg4c, Atg5, UVRAG, Bif-1) involved in the early stages of autophagy, are positive regulators of autophagy (PTEN, p53, DRAM, DAPK1, ARF) or function as both (Beclin1). Accordingly, the inactivation of autophagy genes promotes tumor development in mice and overexpression of some autophagy genes inhibits formation of breast tumors in mice models. Beclin1 is monoallelically deleted in a number of human breast, ovarian, and prostrate cancers. Certain forms of cancer are associated with decreased autophagic activity. In line with these observations protooncogenes like class I PI3K and AKT negatively affect autophagy. Furthermore, autophagy limits necrosis which prevents leukocyte recruitment and permeation of the tumor site. Conversely, tumor cells are often dependent on the prosurvival function of autophagy especially during the later stages of cancer progression.

Autophagy has also been shown to act as a tumor promoter. An upregulation in autophagy triggered by the harsh conditions usually present during tumorigenesis such as increased metabolic demands, hypoxia, genotoxic stress, and hormone deprivation, enables tumor cells to survive and further enable them to resist stress conditions like chemotherapy and radiation treatment. During metastasis, energy conservation and a state of dormancy conferred by autophagy to sustain the cells during this time of stress and starvation is more beneficial than cell proliferation and protein synthesis. In apoptosis-defective bone marrow cancer cells, autophagy induction enhances cell survival and autophagy inhibition accelerates cell death. In RAS-driven cancers, upregulated autophagy allows tumor cells to meet the heightened requirements for nutrients and building blocks required for cell proliferation.

Modulation of Autophagy for Cancer Treatment

A cell death and survival functions of autophagy do not exclude each other and it is clear from morphological and genetic data that a complex interplay exists between autophagy and apoptosis. Cell death can arise from high levels of autophagy though self-digestion. Low levels of autophagy might contribute to cell death by making the cell more susceptible to environmental stressors that are otherwise eliminated by autophagy. Additionally, autophagy has been observed to accompany apoptosis in the facilitation of programmed cell death.

Autophagy can be a tumor suppressive mechanism for normal cells to maintain organelle function, reduce inflammation, DNA damage, genetic instability, degrade aggregated proteins, and maintain homeostasis in the cell, and be used as a mechanism for cell death; however, once tumor cells have transformed and become malignant, autophagy is hijacked into a mechanism that promotes survival of the tumor cell by providing much needed components for rapid cell proliferation, maintenance of functional mitochondria and reduction of oxidative and other forms of stress.

A variety of cancer cell lines and mouse models have been used to try to elucidate the effects of autophagy modulation on tumor initiation and progression. Autophagy inactivation has shown real promise as an effective therapeutic strategy. In KRAS-activated mouse models of non-small-cell lung cancer, genetic deletion of a key autophagy protein caused tumors to stop growing and undergo cell death and also reverted malignant adenomas and carcinomas into benign oncocytomas. In mouse models of RAS-driven pancreatic cancer, inhibition of autophagy reduced tumor growth. Pharmacological inhibition of autophagy using the anti-malaria drug hydroxychloroquine (HCQ) was suppressed survival and tumor growth in human pancreatic cell cancer lines and transplanted human pancreatic cancers. Chloroquinine (CQ) and HCQ block autophagy at a late stage by preventing acidification of the lysosome thus disrupting degradation of autophagosomes and their cargo. Inhibition of autophagy also sensitized melanomas to leucine deprivation.

Since autophagy also enables tumor cells to survive chemotherapy and radiation treatments, supplementing these treatments with the inhibition of autophagy could enhance their efficacy in killing cancer cells. This strategy of combining autophagy inhibition with another anticancer therapy has proven effective for a variety of tumor models which include glioma, myeloma, breast, colon, and prostate cancers. Chronic myelogenous leukemia (CML) can be treated with imatinib (Gleevac), an inducer of autophgy. The efficiency of killing CML progenitor cells in not so efficient with imatinib alone, however when used in combination with the autophagy inhibitor CQ, CML stem cells are almost completely eliminated in vitro. RNAi against atg5 and atg7, essential autophagy genes, inhibited autophagy and sensitized CML cells to undergo cell death. CQ cotreatment with tamoxifen has also been employed to augment p53-mediated apoptosis in a Myc-driven B cell lymphoma model. CQ together with cyclophosphamide, a lymphoma drug, delayed tumor recurrence. HCQ was used in combination chemotherapy with BRAF inhibitor resulting in significant reduction of the mouse xenograft melanoma tumors. This results of this combination was also confirmed in vitro and in vivo in pediatric gliomas. This evidence is further supporting autophagy suppression as a useful cotreatment strategy for fighting cancer. In some cases, autophagy inhibition has been utilized to overcome drug resistance. Currently, there are many ongoing clinical trials in various stages which use autophagy suppression to complement a primary treatment.

Further study is required to fully understand the effects of autophagy on cell death, its ability to play both pro- and antiapoptotic roles in the same cell depending on the death stimulus and the stage of autophagy affected. Furthermore, most drugs administered to inhibit autophagy are not specific for autophagy and therefore other targets in other processes and pathways may be affected. The development of autophagy-specific inhibitors would aid in elucidating the role of autophagy during the various steps of autophagy, triggers of cell death, and stages of cancer development and progression. Many clinical trials are currently underway that use autophagy inhibition as a strategy for cancer therapy. A synergistic approach that involves the modulation of autophagy in combination with other drugs and treatments which trigger cell death could lead to effective treatment options for many types of cancer and appears to be a promising approach in the battle against cancer.

It is now clear that autophagy and cancer are intimately linked. But it is also becoming evident that autophagy, being a homeostatic as well as a stress-inducible cellular process, is regulated in a highly complex way in health and disease.

Cross-References