Autophagy in the pathogenesis of myelodysplastic syndrome and acute myeloid leukemia

Autophagy is a conserved cellular pathway responsible for the sequestration of spent organelles and protein aggregates from the cytoplasm and their delivery into lysosomes for degradation. Autophagy plays an important role in adaptation to starvation, in cell survival, immunity, development and cancer. Recent evidence in mice suggests that autophagic defects in hematopoietic stem cells (HSCs) may be implicated in leukemia. Indeed, mice lacking Atg7 in HSCs develop an atypical myeloproliferation resembling human myelodysplastic syndrome (MDS) progressing to acute myeloid leukemia (AML). Our studies suggest that accumulation of damaged mitochondria and reactive oxygen species result in cell death of the majority of progenitor cells and, possibly, concomitant transformation of some surviving ones. Interestingly, bone marrow cells from MDS patients are characterized by mitochondrial abnormalities and increased cell death. A role for autophagy in the transformation to cancer has been proposed in other cancer types. This review focuses on autophagy in human MDS development and progression to AML within the context of the role of mitochondria, apoptosis and reactive oxygen species (ROS) in its pathogenesis.Key words: autophagy, mitophagy, Atg7, hematopoiesis, HSCs, myelodysplastic syndrome, acute myeloid leukemia     

Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation

The regulated lysosomal degradation pathway of autophagy prevents cellular damage and thus protects from malignant transformation. Autophagy is also required for the maturation of various hematopoietic lineages, namely the erythroid and lymphoid ones, yet its role in adult hematopoietic stem cells (HSCs) remained unexplored. While normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs or early progenitors leads to leukemia. Mechanisms protecting HSCs from cellular damage are therefore essential to prevent hematopoietic malignancies. By conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system, we found that autophagy is required for the maintenance of true HSCs and therefore also of downstream hematopoietic progenitors. Loss of autophagy in HSCs leads to the expansion of a progenitor cell population in the bone marrow, giving rise to a severe, invasive myeloproliferation, which strongly resembles human acute myeloid leukemia (AML).     

Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation

The regulated lysosomal degradation pathway of autophagy prevents cellular damage and thus protects from malignant transformation. Autophagy is also required for the maturation of various hematopoietic lineages, namely the erythroid and lymphoid ones, yet its role in adult hematopoietic stem cells (HSCs) remained unexplored. While normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs or early progenitors leads to leukemia. Mechanisms protecting HSCs from cellular damage are therefore essential to prevent hematopoietic malignancies. By conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system, we found that autophagy is required for the maintenance of true HSCs and therefore also of downstream hematopoietic progenitors. Loss of autophagy in HSCs leads to the expansion of a progenitor cell population in the bone marrow, giving rise to a severe, invasive myeloproliferation, which strongly resembles human acute myeloid leukemia (AML).     

FIP200, an essential component of mammalian autophagy is indispensible for fetal hematopoiesis

Autophagy, an evolutionarily conserved cellular process for bulk protein degradation through lysosomes, plays important roles in various physiological and pathological processes. Recent studies suggest that autophagy also participates in erythroid development. However, to what extent autophagy is involved in hematopoiesis is largely unknown. FIP200 (focal adhesion kinase family interacting protein of 200 kD) is a newly identified essential autophagy gene and a component of the ULK-Atg13-FIP200 complex. We show that mice lacking FIP200 in hematopoietic cells (CKO mice) experience perinatal lethality associated with severe erythroblastic anemia. FIP200 is cell-autonomously required for the maintenance and function of fetal hematopoietic stem cells (HSCs). FIP200 deletion in HSCs does not result in increased apoptosis. However, aberrantly increased HSC proliferation and myeloid expansion are found in CKO embryos, which may be responsible for the depletion of fetal HSCs. Consistent with an essential role of FIP200 in autophagy, FIP200-null fetal HSCs as well as other hematopoietic cells exhibit increased mitochondria mass and reactive oxygen species (ROS). Together, our data identify FIP200 as a key intrinsic regulator of fetal HSCs and suggest a role of autophagy in fetal hematopoiesis and the maintenance of fetal HSCs.     

FIP200, an essential component of mammalian autophagy is indispensible for fetal hematopoiesis

Autophagy, an evolutionarily conserved cellular process for bulk protein degradation through lysosomes, plays important roles in various physiological and pathological processes. Recent studies suggest that autophagy also participates in erythroid development. However, to what extent autophagy is involved in hematopoiesis is largely unknown. FIP200 (focal adhesion kinase family interacting protein of 200 kD) is a newly identified essential autophagy gene and a component of the ULK-Atg13-FIP200 complex. We show that mice lacking FIP200 in hematopoietic cells (CKO mice) experience perinatal lethality associated with severe erythroblastic anemia. FIP200 is cell-autonomously required for the maintenance and function of fetal hematopoietic stem cells (HSCs). FIP200 deletion in HSCs does not result in increased apoptosis. However, aberrantly increased HSC proliferation and myeloid expansion are found in CKO embryos, which may be responsible for the depletion of fetal HSCs. Consistent with an essential role of FIP200 in autophagy, FIP200-null fetal HSCs as well as other hematopoietic cells exhibit increased mitochondria mass and reactive oxygen species (ROS). Together, our data identify FIP200 as a key intrinsic regulator of fetal HSCs and suggest a role of autophagy in fetal hematopoiesis and the maintenance of fetal HSCs.     

Triazole analog 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol induces reactive oxygen species and autophagy-dependent apoptosis in both in vitro and in vivo breast cancer models

Autophagy is considered as an important cell death mechanism that closely interacts with other common cell death programs like apoptosis. Critical role of autophagy in cell death makes it a promising, yet challenging therapeutic target for cancer. We identified a series of 1,2,3-triazole analogs having significant breast cancer inhibition property. Therefore, we attempted to study whether autophagy and apoptosis were involved in the process of cancer cell inhibition. The lead molecule, 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol (T-12) induced significant cell cycle arrest, mitochondrial membrane depolarization, apoptosis and autophagy in MCF-7 and MDA-MB-231 cells. T-12 increased reactive oxygen species and its inhibition by N-acetyl-l-cysteine protected breast cancer cells from autophagy and apoptosis. Autophagy inhibitor, 3-methyladenine abolished T-12 induced apoptosis, mitochondrial membrane depolarization and reactive oxygen species generation. This suggested that T-12 induced autophagy facilitated cell death rather than cell survival. Pan-caspase inhibition did not abrogate T-12 induced autophagy, suggesting that autophagy precedes apoptosis. In addition, T-12 inhibited cell survival pathway signaling proteins, Akt, mTOR and Erk1/2. T-12 also induced significant regression of tumor with oral dose of as low as 10 mg/kg bodyweight in rat mammary tumor model without any apparent toxicity. In presence of reactive oxygen species inhibitor (N-acetyl-l-cysteine) and autophagy inhibitor (chloroquine), T-12 induced tumor regression was significantly decreased. In conclusion, T-12 is a potent inducer of autophagy-dependent apoptosis in breast cancer cells both in vitro and in vivo and can serve as an important lead in development of new anti-tumor therapy.     

ROS, mitochondria and the regulation of autophagy

Accumulation of reactive oxygen species (ROS) is an oxidative stress to which cells respond by activating various defense mechanisms or, finally, by dying. At low levels, however, ROS act as signaling molecules in various intracellular processes. Autophagy, a process by which eukaryotic cells degrade and recycle macromolecules and organelles, has an important role in the cellular response to oxidative stress. Here, we review recent reports suggesting a regulatory role for ROS of mitochondrial origin as signaling molecules in autophagy, leading, under different circumstances, to either survival or cell death. We then discuss the relationship between mitochondria and autophagosomes and propose that mitochondria have an essential role in autophagosome biogenesis.     

Mitochondrial ROS generation for regulation of autophagic pathways in cancer

Mitochondria, the main source of reactive oxygen species (ROS), are required for cell survival; yet also orchestrate programmed cell death (PCD), referring to apoptosis and autophagy. Autophagy is an evolutionarily conserved lysosomal degradation process implicated in a wide range of pathological processes, most notably cancer. Accumulating evidence has recently revealed that mitochondria may generate massive ROS that play the essential role for autophagy regulation, and thus sealing the fate of cancer cell. In this review, we summarize mitochondrial function and ROS generation, and also highlight ROS-modulated core autophagic pathways involved in ATG4–ATG8/LC3, Beclin-1, p53, PTEN, PI3K–Akt–mTOR and MAPK signaling in cancer. Therefore, a better understanding of the intricate relationships between mitochondrial ROS and autophagy may ultimately allow cancer biologists to harness mitochondrial ROS-mediated autophagic pathways for cancer drug discovery.     

Involvement of autophagy in oncogenic K-Ras-induced malignant cell transformation

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-Ras(V12))-induced malignant cell transformation. Retroviral expression of K-Ras(V12) induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-Ras(V12)-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-Ras(V12). Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-Ras(V12)-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-Ras(V12), and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-Ras(V12)-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.     

Lipid oxidation and autophagy in yeast

Autophagy, a process involved in the degradation and the recycling of long-lived proteins and organelles to survive nitrogen starvation, is generally non-selective. However, recent data suggest that selective forms of autophagy exist, that are able to specifically target several organelles, including mitochondria. Conversely, mitochondrial alterations could trigger autophagy. Such a selective form of autophagy might be involved in the elimination of damaged mitochondria. We reported previously that, mitochondria were early targets of rapamycin-induced autophagy. Here we report that rapamycin-induced autophagy is accompanied by the early production of reactive oxygen species and by the early oxidation of mitochondrial lipid. Inhibition of these oxidative effects by resveratrol largely impaired autophagy of both cytosolic proteins and mitochondria, and delayed subsequent cell death. These results support a role of mitochondrial oxidation events in the activation of autophagy.     

Hoechst 33342-induced autophagy protected HeLa cells from caspase-independent cell death with the participation of ROS

Autophagy, an evolutionarily-conserved intracellular organelle and protein degradation process, may exhibit drastically different effects on cell survival depending on the particular environmental and culturing conditions. Hoechst 33342 (HO), a fluorescent dye widely used for staining DNA, has been reported to induce apoptosis in mammalian cells. Here we showed that, in addition to caspase-independent cell death, HO also induced autophagy in HeLa cells, as evidenced by the accumulation of autophagosomes, LC3 form conversion and LC3 puncta formation in a cell line stably expressing GFP-LC3. HO treatment led to generation of reactive oxygen species (ROS), and inhibition of ROS with N-acetyl-l-cysteine (NAC) abrogated both autophagy and caspase-independent cell death. Finally, autophagy played a protective role against caspase-independent cell death, as cell death induced by HO was enhanced under pharmacological and siRNA-mediated genetic inhibition of autophagy.     

Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells

Autophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H(2)O(2)) and 2-methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H(2)O(2) or 2-ME-induced cell death. H(2)O(2) and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H(2)O(2) or 2-ME through overexpression of manganese-superoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H(2)O(2)- or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H(2)O(2) or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in non-transformed cells.     

Induction of Reactive Oxygen Species-mediated Autophagy by a Novel Microtubule-modulating Agent

Autophagy is being increasingly implicated in both cell survival and death. However, the intricate relationships between drug-induced autophagy and apoptosis remain elusive. Here we demonstrate that a tubulin-binding noscapine analog, (R)-9-bromo-5-((S)-4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]-di-oxolo[4,5-g]isoquinoline (Red-Br-nos), exerts a novel autophagic response followed by apoptotic cell death in human prostate cancer PC-3 cells. Red-Br-nos-induced autophagy was an early event detectable within 12 h that displayed a wide array of characteristic features including double membranous vacuoles with entrapped organelles, acidic vesicular organelles, and increased expression of LC3-II and beclin-1. Red-Br-nos-triggered release of reactive oxygen species (ROS) and attenuation of ROS by tiron, a ROS scavenger, reduced the sub-G₁ population suggesting ROS-dependent apoptosis. Abrogation of ROS also reduced autophagy indicating that ROS triggers autophagy. Pharmacological and genetic approaches to inhibit autophagy uncovered the protective role of Red-Br-nos-induced autophagy in PC-3 cells. Direct effects of the drug on mitochondria viz. disruption of normal cristae architecture and dissipation of mitochondrial transmembrane potential revealed a functional link between ROS generation, autophagy, and apoptosis induction. This is the first report to demonstrate the protective role of ROS-mediated autophagy and induction of caspase-independent ROS-dependent apoptosis in PC-3 cells by Red-Br-nos, a member of the noscapinoid family of microtubule-modulating anticancer agents.     

The role of autophagy in mitochondria maintenance: characterization of mitochondrial functions in autophagy-deficient S. cerevisiae strains

Autophagy is a lysosome-dependent cellular degradation process. Organisms bearing deletions of the essential autophagy genes exhibit various pathological conditions, including cancer in mammals and shortened life span in C. elegans. The direct cause forthese phenotypes is not clear. Here we used yeast as a model system to characterize the cellular consequence of ATG (autophagy-related) gene deletions. We found that the atgmutant strains, atg1delta, atg6delta, atg8delta and atg12delta, showed defects related to mitochondrial biology. These strains were unable to degrade mitochondria in stationary culture. In non-fermentable medium, which requires mitochondrial oxidative phosphorylation for survival, these atg strains showed a growth defect with an increased cell population at the G(1) phase of the cell cycle. The cells had lower oxygen consumption rates and reduced mitochondrial electron transport chain activities. Under these growth conditions, the atg strains had lower mitochondrial membrane potential. In addition, these mutants generated higher levels of reactive oxygen species (ROS) and they were prone to accumulate dysfunctional mitochondria. This study clearly indicates that an autophagy defect has a functional impact on various aspects of mitochondrial functions and suggests a critical role of autophagy in mitochondria maintenance.     

Platinum‐based combination chemotherapy triggers cancer cell death through induction of BNIP3 and ROS,but not autophagy

These days, cancer can still not be effectively cured because cancer cells readily develop resistance to anticancer drugs. Therefore, an effective combination of drugs with different mechanisms to prevent drug resistance has become a very important issue. Furthermore, the BH3‐only protein BNIP3 is involved in both apoptotic and autophagic cell death. In this study, lung cancer cells were treated with a chemotherapy drug alone or in combination to identify the role of BNIP3 and autophagy in combination chemotherapy for treating cancer. Our data revealed that various combinational treatments of two drugs could increase cancer cell death and cisplatin in combination with rapamycin or LBH589, which triggered the cell cycle arrest at the S phase. Cells with autophagosome and pEGFP‐LC3 puncta increased when treated with drugs. To confirm the role of autophagy, cancer cells were pre‐treated with the autophagy inhibitor 3‐methyladenine (3‐MA). 3‐MA sensitized cancer cells to chemotherapy drug treatments. These results suggest that autophagy may be responsible for cell survival in combination chemotherapy for lung cancer. Moreover, BNIP3 was induced and localized in mitochondria when cells were treated with drugs. The transfection of a dominant negative transmembrane deletion construct of BNIP3 (BNIP3ΔTM) and treatment of a reactive oxygen species (ROS) inhibitor suppressed chemo drug‐induced cell death. These results indicate that BNIP3 and ROS may be involved in combination chemo drug‐induced cell death. However, chemo drug‐induced autophagy may protect cancer cells from drug cytotoxicity. As a result, inhibiting autophagy may improve the effects of combination chemotherapy when treating lung cancer.     

Hoechst 33342-induced autophagy protected HeLa cells from caspase-independent cell death with the participation of ROS

Abstract

Autophagy, an evolutionarily-conserved intracellular organelle and protein degradation process, may exhibit drastically different effects on cell survival depending on the particular environmental and culturing conditions. Hoechst 33342 (HO), a fluorescent dye widely used for staining DNA, has been reported to induce apoptosis in mammalian cells. Here we showed that, in addition to caspase-independent cell death, HO also induced autophagy in HeLa cells, as evidenced by the accumulation of autophagosomes, LC3 form conversion and LC3 puncta formation in a cell line stably expressing GFP-LC3. HO treatment led to generation of reactive oxygen species (ROS), and inhibition of ROS with N-acetyl-l-cysteine (NAC) abrogated both autophagy and caspase-independent cell death. Finally, autophagy played a protective role against caspase-independent cell death, as cell death induced by HO was enhanced under pharmacological and siRNA-mediated genetic inhibition of autophagy.     

Autophagy

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.     

Autophagy: a cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.     

P38 MAP kinase functions as a switch in MS-275-induced reactive oxygen species-dependent autophagy and apoptosis in human colon cancer cells

MS-275 is a synthetic benzamide derivative of the histone deacetylase inhibitor and is currently in phase I/II clinical trials. Many reports have shown that the anti-tumor activity of MS-275 in several types of cancer is mainly attributable to its capacity to induce the apoptotic death of tumor cells. It remains unclear if autophagy is involved in MS-275 treatment of cancer cells. Here, we first show that MS-275 induces human colon cancer cell HCT116 autophagy as well as apoptosis. Short-term treatment (24h) induced HCT116 cells to undergo autophagy with dependence on intracellular reactive oxygen species production and ERK activation. The activated reactive oxygen species/ERK signal promoted Atg7 protein expression, which triggered MS-275-induced cancer cell autophagy. However, after prolonged treatment with MS-275 (over 48h), autophagic cells turned apoptotic, which was also dependent on reactive oxygen species generation. Interestingly, we found that p38 MAP kinase played a vital role in the switch from autophagy to apoptosis in MS-275-induced human colon cancer cells. High expression of p38 induced cell autophagy, but low expression resulted in apoptosis. In addition, observations in vivo are strongly consistent with the in vitro results. Therefore, these findings extend our understanding of the action of MS-275 in inducing cancer cell death and suggest that it may be a promising clinical chemotherapeutic agent with multiple effects.     

Oxidative stress induces autophagy in response to multiple noxious stimuli in retinal ganglion cells

Retinal ganglion cells (RGCs) are the only afferent neurons that can transmit visual information to the brain. The death of RGCs occurs in the early stages of glaucoma, diabetic retinopathy, and many other retinal diseases. Autophagy is a highly conserved lysosomal pathway, which is crucial for maintaining cellular homeostasis and cell survival under stressful conditions. Research has established that autophagy exists in RGCs after increasing intraocular pressure (IOP), retinal ischemia, optic nerve transection (ONT), axotomy, or optic nerve crush. However, the mechanism responsible for defining how autophagy is induced in RGCs has not been elucidated. Accumulating data has pointed to an essential role of reactive oxygen species (ROS) in the activation of autophagy. RGCs have long axons with comparatively high densities of mitochondria. This makes them more sensitive to energy deficiency and vulnerable to oxidative stress. In this review, we explore the role of oxidative stress in the activation of autophagy in RGCs, and discuss the possible mechanisms that are involved in this process. We aim to provide a more theoretical basis of oxidative stress-induced autophagy, and provide innovative targets for therapeutic intervention in retinopathy.