Cancer cell death : the interplay of autophagy and apoptosis Molecular genetics Lab Hyun Hwang-Bo

Context 1. Introduction Autophagy Apoptosis 2. Interplay of autophagy and apoptosis Signals that induce both apoptosis and autophagy Effects of autophagy on lethal signaling Impact of autophagy on the removal of dead cells in tissues 3. Conclusion

Introduction

Autophagy Macroautophagy is extensively involved in cellular homeostasis. Macroautophay (hereafter referred to as autophagy) is usually a catabolic process in which portions of the cytoplasm are sequestered within cytosolic double-membrane vesicles called autophagosomes and subsequently delivered to the lysosome to allow degradation and recycling of the cargo. Autophagy has been recently implicated in various human pathological and physiological conditions, such as neurodegeneration, immunity, cancer, development, myopathies, heart diseases, liver diseases and longevity. Basal autophagy is essential for removing misfolded proteins and damaged organelles, and therefore, plays a vital role in maintaining cellular homeostasis in all tissues. My major research interests include understanding the molecular mechanisms regulating mammalian autophagy and identification of small molecule autophagy enhancers as a therapeutic strategy for neurodegenerative diseases. Autophagy is negatively regulated by the mammalian Target Of Rapamycin (mTOR; a serine/threonine kinase), and can be induced by the mTOR inhibitor rapamycin. However, mTOR has many vital cellular functions like translation and cell growth, and therefore, it is highly desirable to discover mTOR-independent, autophagy-inducing pathways/drugs as therapeutic targets. My work involves in the identification of autophagy modulators that regulate autophagy independently of mTOR. Identification of autophagy-inducing drugs has huge therapeutic potential not only for neurodegenerative diseases, but also for other diseases where autophagy acts as a protective pathway.  Dev Cell. 2010 Jul 20;19(1):11-2.

Autophagy Conditions such as nutrient starvation, pathogen infection and other environmental stressors, can induce autophagy. Autophagy begins with the isolation of double-membrane-bound structures inside an intact cell. Autophagy is intracellular lysosomal degradation and recycling of proteins and organelles. Autophagy-related genes (Atg) control the process of autophagy (1). The products of these Atg genes are regulated by nutrient (mTOR), energy [AMP-activated protein (AMPK)], and stress [hypoxia-inducible factors (HIF)] sensing mechanisms in the cell that turn the pathway on and off (Fig. 1). Once activated, a series of ATG protein complexes orchestrate the formation of double membrane vesicles called autophagosomes that capture cytoplasmic cargo (Fig. 1). Cargo can be damaged or superfluous proteins, organelles, lipids, and glycogen that are tagged with ubiquitin and recognized by autophagy receptors such as Sequestasome1 (p62). Cargo receptors bind both cargo and the autophagosome membrane component LC3-II, facilitating cargo sequestration. Fusion between autophagosomes and lysosomes provides the hydrolases to degrade the cargo. The resulting amino acids, nucleosides, fatty acids, and sugars are released into the cytoplasm for recycling (ref. 2; Fig. 1). Autophagy is essential to prevent the toxic accumulation of damaged proteins and organelles and to sustain metabolism, energy homeostasis, and survival in starvation. Autophagy is also important to recycle ferritin, and autophagy defects cause perturbation of iron homeostasis that increases susceptibility to oxidative stress (3, 4).. As autophagy activity depends on the level of activation of autophagosome initiation and the rate of cargo degradation in lysosomes, flux through the pathway is critical to assess. This process typically involves blocking lysosome function and measuring the accumulation of autophagolysosomes and autophagy substrates. Alternatively, genetically engineered mouse models (GEMM) of constitutive and conditional knockouts of essential Atg genes provide an accurate assessment of the functional requirement for autophagy. A great deal of what we know about the role of autophagy in normal tissues comes from these models, which also inform its role in cancer. This review focuses on what we know about the proposed tumor suppression and promotion roles for autophagy and how we may apply this knowledge to the treatment and prevention of human cancer. Conditions such as nutrient starvation, pathogen infection and other environmental stressors, can induce autophagy. Autophagy begins with the isolation of double-membrane-bound structures inside an intact cell. Previously, these structures were believed to be derived from the ribosome-free region of the rough endoplasmic reticulum82,83, but recent studies indicate that they might originate from a preexisting membrane structure called a phagophore84, or could be formed de novo80,85. These membrane structures elongate and mature, and microtubule-associated protein 1 light chain 3 (LC3) is recruited to the membrane. The elongated double membranes form autophagosomes, which sequester cytoplasmic proteins and organelles such as mitochondria. The formation of the pre-autophagosomal structure can be inhibited by the phosphatidylinositol 3-phosphate kinase (PI3K) inhibitor 3-methyladenine (3-MA). Sequestration requires ATP and is regulated mainly by class III PI3K46. The autophagosomes mature with acidification by the H+-ATPase71 and fuse with lysosomes to become autolysosomes (also known as the degradative autophagic vacuoles). Microtubules are important mediators of this fusion process. This process is inhibited by the H+-ATPase inhibitor bafilomycin A1, or by microtubule inhibitors such as vinblastine and nocodazole86,87. Eventually, the sequestered contents are degraded by lysosomal hydrolases for recycling. One assay for autophagic cells is to detect the presence of membrane-bound LC3, which accumulates on autophagosomes. Nat Rev Cancer. 2005 Sep;5(9):726-34. Review.

Autophagy — the basics The core autophagy pathway starts with the formation of an isolation membrane (also called a phagophore), most often at the contact sites between mitochondria and the endoplasmic reticulum Nat Rev Mol Cell Biol. 2014 Feb;15(2):81-94.

Importance of apoptosis And are phagocytosed in vivo Apoptosis describes the orchestrated collapse of a cell characterised by membrane blebbing, cell shrinkage, condensation of chromatin, and fragmentation of DNA followed by rapid engulfment of the corpse by neighbouring cells. It is distinguished from death by necrosis by the absence of an associated inflammatory response. Importance of apoptosis Importance in normal physiology/ development -development: immune systems maturation, morphogenesis, neural development -adult: immune privilege, DNA damage and wound repair Excess apoptosis neurodegenerative diseases Deficient apoptosis cancer and autoimmunity Healthy cell Shrinks and the condensed chromatin collapses into crescents around the nuclear envelope The blebbing increases and the cell finally breaks into a number of apoptotic bodies Ormigita-k.blogspot.com A cell initiates intracellular apoptotic signaling in response to a stress, which may bring about cell suicide. The binding of nuclear receptors by glucocorticoids,[18] heat,[18] radiation,[18] nutrient deprivation,[18] viral infection,[18] hypoxia[18] and increased intracellular calcium concentration,[19][20] for example, by damage to the membrane, can all trigger the release of intracellular apoptotic signals by a damaged cell. A number of cellular components, such as poly ADP ribose polymerase, may also help regulate apoptosis.[21] Importance of apoptosis Importance in normal physiology/development -development: immune systems maturation, morphogenesis, neural development -adult: immune privilege, DNA damage and wound repair 2.Excess apoptosis -neurodegenerative diseases 3.Deficient apoptosis -cancer -autoimmunity And are phagocytosed in vivo

Apoptosis Apoptosis is accompanied by a sequence of characteristic biochemical changes, including mitochondrial outer membrane permeabilization (MOMP), activation of the effector caspases caspase 3, caspase 6 and caspase 7, and the activation of catabolic hydrolases that degrade most of the macromolecules of the cell, which includes DNA. Nat Rev Mol Cell Biol. 2014 Feb;15(2):81-94.

Potential strategies for treating cancer by manipulating the autophagic process Evidence indicates that the modulation of autophagy is an important component of tumorigenesis, making it a possible therapeutic target. How might this cellular process be manipulated to improve the treatment of cancer? Nat Rev Cancer. 2005 Sep;5(9):726-34. Review.

Comparison of autophaic cell death and apoptosis When cell death involves autophagy, it is designated as type II PROGRAMMED CELL DEATH (PCD) or autophagic cell death, in contrast to apoptosis, which is referred to as type I. The morphological and biochemical features of autophagic cell death and apoptosis are generally distinct.

Interplay of autophagy and apoptosis

Signals that induce both apoptosis and autophagy. p53. The tumour suppressor p53 exerts both pro- and anti-autophagic functions, which depends on its subcellular localization and activation status. In response to stress, p53 translocates to the nucleus, where it binds to the promoter region of multiple pro-autophagic genes, including the β1, β2 and γ‑subunits of AMP-activated protein kinase (AMPK), damage-regulated autophagy modulator 1 (DRAM1), sestrin 1, sestrin 2 and phosphatase and tensin homologue (PTEN). FIP200 (FAK family kinase-interacting protein of 200 kDa) ULK1 (UNC-51-like kinase 1) MOMP (mitochondrial outer membrane permeabilization) DRAM (DNA damage-regulated autophagy modulator 1) Nat Rev Mol Cell Biol. 2014 Feb;15(2):81-94.

Signals that induce both apoptosis and autophagy. Through its BH3 domain, Beclin 1 interacts with anti-apoptotic proteins from the BCL‑2 family. This inhibits the pro-autophagic function of Beclin 1, but does not interfere with the anti-apoptotic activity of the BCL‑2 family proteins. BH3‑only proteins, as well as BH3 mimetics, disrupt this interaction, which enables Beclin 1 to increase autophagic activity. BIM interacts with Beclin 1 and mislocalizes it to dynein light chain 1, thereby preventing autophagy. VPS34 (Vacuolar protein sorting 34) NIX (NIP3-like protein X) Nat Rev Mol Cell Biol. 2014 Feb;15(2):81-94.

Signals that induce both apoptosis and autophagy. Death associated protein kinase (DAPK). This kinase can stimulate autophagy by phosphorylating Beclin 1, which enables its dissociation from BCL‑2 and its association with vacuolar protein sorting 34 (VPS34). DAPK can also activate a second kinase, protein kinase D (PKD), which phosphorylates and activates VPS34. DAPK (death-associated protein kinase) JNK (JUN N-terminal kinase) PKD (protein kinase D) Nat Rev Mol Cell Biol. 2014 Feb;15(2):81-94.

Signals that induce both apoptosis and autophagy. JUN N‑terminal kinase (JNK). JNK induces autophagy by phosphorylating BCL‑2 or BIM, which disrupts their inhibitory interaction with Beclin 1. This leads to an active Beclin 1–VPS34 complex and autophagy and also prevents BCL‑2 from inhibiting pro-apoptotic proteins, thus promoting apoptosis. Nat Rev Mol Cell Biol. 2014 Feb;15(2):81-94.

Effects of autophagy on lethal signalling. - Inhibition of apoptosis and anoikis by autophagy One of the principal mechanisms through which autophagy reduces the tendency of cells to undergo apoptosis is mitophagy. Front Physiol. 2013 Dec 26;4:384. Nat Rev Mol Cell Biol. 2014 Feb;15(2):81-94.

Effects of autophagy on lethal signalling. -Role of autophagy proteins in cell death. Autophagy can mediate cell death as an effector mechanism, in a process known as autophagic cell death (ACD). ACD refers to cell death by autophagy in which the specific suppression of autophagy by inhibiting, depleting or deleting several distinct essential autophagy genes and/ or proteins prevents cell death. Mol Cell. 2011 Dec 23;44(6):844-5. Nat Rev Mol Cell Biol. 2014 Feb;15(2):81-94.

Impact of autophagy on the removal of dead cells in tissues. During the course of developmental cell death, pre-mortem autophagy is required for the release of LPC, a potent chemotactic signal, and for the exposure of the ‘eat‑me’ signal PtdSer, on the cell surface. Pre-mortem autophagy induction is essential for ATP release from dying cancer cells. Inhibition of ATP release compromises the recruitment of immune cells, and thus the activation of the immune system. This prevents the immune system from recognizing tumour-specific antigens, a process that is essential for the effectiveness of anti-tumour therapies. LPC (lysophosphatidylcholine) PtdSer (phosphatidylserine) Nat Rev Mol Cell Biol. 2014 Feb;15(2):81-94.

Functional relationship between autophagy and apoptosis. Nat Rev Mol Cell Biol. 2014 Feb;15(2):81-94.

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