Steps Of Autophagy Research Articles

Recent progress in autophagy

Recent progress in autophagy

Autophagy Is Uncoupled From ATG5-Dependent Apoptosis in Cells Resistant To Proteasome Inhibition

Cancer cells face enormous metabolic challenges to meet energetic and biosynthetic demands and, therefore, a hallmark of cancer cells is reprogrammed metabolic circuitry that results from oncogenic events selected during tumorigenesis to promote growth, survival and drug resistance. The increased metabolic demands in tumor cells are offset by autophagy - a basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components and potentially contributes to the growth of cancer cells. The proteasome is the catalytic core of the ubiquitin (Ub)-dependent proteolytic pathway that degrades short-lived and denatured proteins to maintain cell viability. Success of the proteasome inhibitor bortezomib has emerged as standard-of-care therapy for the invariably fatal malignancy MM to catapult the Ub+proteasome system (UPS) into a position of prominence in cancer biology and drug discovery, significant obstacles remain since many patients do not respond to bortezomib and those that do inevitably develop drug resistance through mechanisms that remain elusive. We discovered that in multiple myeloma (MM) cells, bortezomib triggers activation of AMPK - the master regulator of cellular energy metabolism. At physiologically-relevant concentrations, proteasome inhibitors further triggered AMPK-dependent autophagosome formation in myeloma cells that was directly and sequentially linked to apoptosis in drug-sensitive cells. However, in bortezomib-resistant cells, autophagosome formation and apoptosis were uncoupled to enhance drug resistance and the survival of tumor cells. Genetic knockout of the AMPK catalytic subunits in mouse embryonic fibroblasts reduced the effect of bortezomib on both autophagy and apoptosis. Similar effects were seen with genetic ablation of ULK1- a downstream substrate of AMPK - also required for bortezomib-induced autophagosome formation. Enforced expression of the autophagy-related gene Atg5 enhanced bortezomib-induced cell death while bortezomib treatment promoted ATG5 cleavage to yield a truncated, pro-apoptotic form. ATG5 participates in the initial steps of autophagy and was also cleaved by calpain to generate the truncated ATG5 form that translocates to the mitochondria, binds Bcl2 and induces mitochondrial outer membrane permeabilization (MOMP). We propose that ATG5 promotes the displacement of anti-apoptotic Bcl2 proteins from the pro-apoptotic Bcl2 family members, e.g., NOXA and PUMA, to facilitate MOMP - considered the point of commitment to cell death through the mitochondrial apoptotic pathway. Importantly, bortezomib in combination with the AMPK activators AICAR or metformin enhanced ATG5 cleavage and overcame resistance to proteasome inhibitors. The molecular events that regulate the complex interplay between autophagy and apoptosis as determinants of cell fate under physiologic and pathologic conditions have remained poorly understood. Since there is an urgent need to unravel the mechanism(s) of drug resistance and to develop more effective therapies, we propose pharmacologic repositioning of AMPK activators as a promising strategy to enhance the therapeutic efficacy of proteasome inhibitors to overcome drug resistance in the treatment of hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract 3513: Hydroxychoroquine (HCQ) modulates autophagy in melanoma: preliminary results of a phase 0 trial in patients with resectable melanoma.

Abstract Autophagy enables tumors to survive metabolic stress by recycling intracellular components to sustain metabolism. This suggests that inhibiting autophagy with drugs such as HCQ, a lysosomotropic agent that blocks the final step of autophagy when autophagosomes fuse with lysosomes, may promote tumor cell death. In melanoma cell lines and animal models, HCQ administration resulted in inhibition of flux through the autophagy pathway as measured by visualization of increased numbers of autophagic vesicles (AVs) and processing of the autophagosome marker LC3 from LC3-I to LC3-II. We hypothesized that administration of HCQ can modulate autophagy in human melanoma tumors that can be detected by increased autophagic vesicle (AV) formation and by increased LC3-II/I ratio. Methods: To test this hypothesis, we conducted a phase 0 trial in patients with resectable stage III or IV melanoma. Patients who consented underwent pretreatment tumor biopsies, were treated with either 200 or 400 mg bid of HCQ for 14 days, and then underwent surgery. Paired tumor samples were processed for electron microscopy (EM) and scored by counting AVs per high powered field in multiple fields. Changes in the LC3II/I ratio were examined by Western blot. Quantitation of HCQ in blood was performed using HPLC-fluorescence method in whole blood, plasma and ultrafiltrates. Changes in AV formation on EM, LC3 II/I ratio, and pharmacokinetic (PK) data from the paired observations were to be correlated. Otherwise, the statistics utilized were descriptive. Results: 12 of 14 patients enrolled had tumor sufficient for evaluation. 8 patients were enrolled at 200 mg bid and 4 at 400 mg bid. No significant toxicities were observed. Autophagy modulation was observed in 4 of the 12 tumors as evidenced by increased LC3II/I ratio on Western blot. Observed changes in AV formation correlated with pre-post changes in LC3II/I ratio (correlation r = 0.42, p = 0.20) in this small sample size. HCQ concentrations (ssmin) showed dose proportional increases in blood, plasma and ultrafiltrate. No correlations were evident between changes on EM and HCQ concentration in plasma, whole blood, or plasma ultrafiltrates. Analyses for common driver mutations in melanoma are ongoing. Conclusions: Treatment with HCQ for 14 days resulted in increased LC3II/I ratio indicative of autophagy modulation in 1/3 of tumors from patients with resectable stage III or IV melanoma. The correlation between pre-post changes in AV formation and LC3II/I ratio suggests that LC3II/I ratio may be a useful measurement of autophagy in human tumors, as in the laboratory. The degree of modulation after 14 days of therapy did not appear to be dose or exposure dependent, although HCQ levels had likely not reached steady state at 14 days. Enrollment continues at 600 mg bid to assess if higher doses produce saturable PK, increased AV formation, and stronger correlations among them. Citation Format: Janice M. Mehnert, Xiaoqi Xie, Megha Rajpal, Liesel Dudek, Monal Mehta, Vassiliki Karantza, Murugesan Gounder, Joseph Aisner, Weichung Shih, Rajesh Patel, Chunxia Chen, James Goydos, Ravi K. Amaravadi, Eileen P. White. Hydroxychoroquine (HCQ) modulates autophagy in melanoma: preliminary results of a phase 0 trial in patients with resectable melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3513. doi:10.1158/1538-7445.AM2013-3513

The thiazole derivative CPTH6 impairs autophagy

We have previously demonstrated that the thiazole derivative 3-methylcyclopentylidene-[4-(4′-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) induces apoptosis and cell cycle arrest in human leukemia cells. The aim of this study was to evaluate whether CPTH6 is able to affect autophagy. By using several human tumor cell lines with different origins we demonstrated that CPTH6 treatment induced, in a dose-dependent manner, a significant increase in autophagic features, as imaged by electron microscopy, immunoblotting analysis of membrane-bound form of microtubule-associated protein 1 light chain 3 (LC3B-II) levels and by appearance of typical LC3B-II-associated autophagosomal puncta. To gain insights into the molecular mechanisms of elevated markers of autophagy induced by CPTH6 treatment, we silenced the expression of several proteins acting at different steps of autophagy. We found that the effect of CPTH6 on autophagy developed through a noncanonical mechanism that did not require beclin-1-dependent nucleation, but involved Atg-7-mediated elongation of autophagosomal membranes. Strikingly, a combined treatment of CPTH6 with late-stage autophagy inhibitors, such as chloroquine and bafilomycin A1, demonstrates that under basal condition CPTH6 reduces autophagosome turnover through an impairment of their degradation pathway, rather than enhancing autophagosome formation, as confirmed by immunofluorescence experiments. According to these results, CPTH6-induced enhancement of autophagy substrate p62 and NBR1 protein levels confirms a blockage of autophagic cargo degradation. In addition, CPTH6 inhibited autophagosome maturation and compounds having high structural similarities with CPTH6 produced similar effects on the autophagic pathway. Finally, the evidence that CPTH6 treatment decreased α-tubulin acetylation and failed to increase autophagic markers in cells in which acetyltransferase ATAT1 expression was silenced indicates a possible role of α-tubulin acetylation in CPTH6-induced alteration in autophagy. Overall, CPTH6 could be a valuable agent for the treatment of cancer and should be further studied as a possible antineoplastic agent.

ZKSCAN3 Is a Master Transcriptional Repressor of Autophagy

Autophagy constitutes a major cell-protective mechanism that eliminates damaged components and maintains energy homeostasis via recycling nutrients under normal/stressed conditions. Although the core components of autophagy have been well studied, regulation of autophagy at the transcriptional level is poorly understood. Herein, we establish ZKSCAN3, a zinc finger family DNA-binding protein, as a transcriptional repressor of autophagy. Silencing of ZKSCAN3 induced autophagy and increased lysosome biogenesis. Importantly, we show that ZKSCAN3 represses transcription of a large gene set (>60) integral to, or regulatory for, autophagy and lysosome biogenesis/function and that a subset of these genes, including Map1lC3b and Wipi2, represent direct targets. Interestingly, ZKSCAN3 and TFEB are oppositely regulated by starvation and in turn oppositely regulate lysosomal biogenesis and autophagy, suggesting that they act in conjunction. Altogether, our study uncovers an autophagy master switch regulating the expression of a transcriptional network of genes integral to autophagy and lysosome biogenesis/function.

Preclinical evaluation of statins as a treatment for ovarian cancer

ObjectiveTo evaluate the potential for statins to treat ovarian cancer. MethodsThe sensitivity of 7 ovarian cancer cell lines to either statins or statins combined with either carboplatin or paclitaxel was assessed using monolayer cultures. Sensitivity to simvastatin was also evaluated in ovarian cancer spheroids. The kinetics of cell death induced by simvastatin was evaluated by measuring Trypan Blue exclusion. Autophagy induced by simvastatin was assessed by measuring LC3-II, p62 or Rab7 by immunoblotting or immunocytochemistry. ResultsAll statins except pravastatin demonstrated single agent activity against monolayers (IC50=1–35μM) and spheroids (IC50=1–13μM). This was mediated by HMG-CoAR inhibition, because either mevalonate or geranylgeraniol prevented the cytotoxic effects of simvastatin. Continuous exposure for 4days was necessary to cause cell death. Simvastatin caused accumulation of p62 but loss of Rab7, suggesting inhibition of autophagosome trafficking. Accumulation of LC3-II was also observed, even in the presence of bafilomycin, suggesting additional stimulation of an earlier step in autophagy. Knockdown of the key autophagy regulator Atg5 caused a modest increase in the sensitivity of Ovcar-8 cells to simvastatin. Finally, additive or mild antagonist effects were observed when simvastatin was combined simultaneously with either carboplatin or paclitaxel, but when cells were exposed to simvastatin prior to carboplatin, profound antagonism was observed. ConclusionsThese observations suggest that clinical trials of statins in ovarian cancer should evaluate high doses and schedules that ensure continual inhibition of HMG-CoAR. Simvastatin has conflicting effects on the autophagy pathway and this may contribute to its cytotoxic activity.

Phase I/II Trial of Autophagy Inhibition in Combination with Neoadjuvant Gemcitabine in High Risk Pancreatic Adenocarcinoma: Safety and Response to Treatment

Autophagy is a mechanism of cell survival in response to cellular stress in which organelles and proteins are recycled for energy production. Autophagy plays a critical role in resistance to therapy in pancreatic cancer and correlates adversely with prognosis. Hydroxychloroquine (HCQ), a commonly prescribed antimalarial drug, blocks acidification of the lysosome, inhibiting the last step in autophagy. Murine models demonstrate that CQ has significant anti-tumor activity and enhances cytotoxic effects of chemotherapy, suggesting a novel treatment strategy in patients with pancreatic cancer.

Resveratrol Reverses Remodeling in Hearts with Large, Old Myocardial Infarctions through Enhanced Autophagy-Activating AMP Kinase Pathway

We investigated the effect of resveratrol, a popular natural polyphenolic compound with antioxidant and proautophagic actions, on postinfarction heart failure. Myocardial infarction was induced in mice by left coronary artery ligation. Four weeks postinfarction, when heart failure was established, the surviving mice were started on 2-week treatments with one of the following: vehicle, low- or high-dose resveratrol (5 or 50 mg/kg/day, respectively), chloroquine (an autophagy inhibitor), or high-dose resveratrol plus chloroquine. High-dose resveratrol partially reversed left ventricular dilation (reverse remodeling) and significantly improved cardiac function. Autophagy was augmented in those hearts, as indicated by up-regulation of myocardial microtubule-associated protein-1 light chain 3-II, ATP content, and autophagic vacuoles. The activities of AMP-activated protein kinase and silent information regulator-1 were enhanced in hearts treated with resveratrol, whereas Akt activity and manganese superoxide dismutase expression were unchanged, and the activities of mammalian target of rapamycin and p70 S6 kinase were suppressed. Chloroquine elicited opposite results, including exacerbation of cardiac remodeling associated with a reduction in autophagic activity. When resveratrol and chloroquine were administered together, the effects offset one another. In vitro, compound C (AMP-activated protein kinase inhibitor) suppressed resveratrol-induced autophagy in cardiomyocytes, but did not affect the events evoked by chloroquine. In conclusion, resveratrol is a beneficial pharmacological tool that augments autophagy to bring about reverse remodeling in the postinfarction heart.

The small GTPases Rab9A and Rab23 function at distinct steps in autophagy during Group A Streptococcus infection

Autophagy mediates the degradation of cytoplasmic contents in the lysosome and plays a significant role in immunity. Here we identified the small GTPases Rab9A and Rab23 as novel autophagy regulators during Group A streptococcus (GAS) infection. Rab9A was recruited to GAS-containing autophagosome-like vacuoles (GcAVs) after autophagosomal maturation and its activity was required for GcAV enlargement and eventual lysosomal fusion. GcAV enlargement appeared to be related to homotypic fusion of GcAVs with Rab9A. Rab23 was recruited to GAS-capturing forming autophagosomes. Knockdown of Rab23 expression decreased both LC3- and Atg5-positive GAS formation and caused the accumulation of LC3-positive structures that did not associate with intracellular GAS. It was suggested, therefore, that Rab23 is required for GcAV formation and is involved in GAS targeting of autophagic vacuoles. Furthermore, knockdown of Rab9A or Rab23 expression impaired the degradation of intracellular GAS. Therefore, our data reveal that the Rab9A and Rab23 GTPases play crucial roles in autophagy of GAS. However, neither Rab9A nor Rab23 were localized to starvation-induced autophagosomes. Not only Rab9A but also Rab23 was dispensable for starvation-induced autophagosome formation. These findings demonstrate that specific Rab proteins function at distinct steps during autophagy in response to GAS infection.

Discovery of Novel DENN Proteins: Implications for the Evolution of Eukaryotic Intracellular Membrane Structures and Human Disease.

The tripartite DENN module, comprised of a N-terminal longin domain, followed by DENN, and d-DENN domains, is a GDP-GTP exchange factor (GEFs) for Rab GTPases, which are regulators of practically all membrane trafficking events in eukaryotes. Using sequence and structure analysis we identify multiple novel homologs of the DENN module, many of which can be traced back to the ancestral eukaryote. These findings provide unexpected leads regarding key cellular processes such as autophagy, vesicle-vacuole interactions, chromosome segregation, and human disease. Of these, SMCR8, the folliculin interacting protein-1 and 2 (FNIP1 and FNIP2), nitrogen permease regulator 2 (NPR2), and NPR3 are proposed to function in recruiting Rab GTPases during different steps of autophagy, fusion of autophagosomes with the vacuole and regulation of cellular metabolism. Another novel DENN protein identified in this study is C9ORF72; expansions of the hexanucleotide GGGGCC in its first intron have been recently implicated in amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). While this mutation is proposed to cause a RNA-level defect, the identification of C9ORF72 as a potential DENN-type GEF raises the possibility that at least part of the pathology might relate to a specific Rab-dependent vesicular trafficking process, as has been observed in the case of some other neurological conditions with similar phenotypes. We present evidence that the longin domain, such as those found in the DENN module, are likely to have been ultimately derived from the related domains found in prokaryotic GTPase-activating proteins of MglA-like GTPases. Thus, the origin of the longin domains from this ancient GTPase-interacting domain, concomitant with the radiation of GTPases, especially of the Rab clade, played an important role in the dynamics of eukaryotic intracellular membrane systems.

Temporal orchestration of circadian autophagy rhythm by C/EBPβ

Temporal organization of tissue metabolism is important for maintaining nutrient and energy homeostasis in mammals. Autophagy is a conserved cellular pathway that is activated in response to nutrient limitation, resulting in the degradation of cytoplasmic components and the release of amino acids and other nutrients. Here, we show that autophagy exhibits robust circadian rhythm in mouse liver, which is accompanied by cyclic induction of genes involved in various steps of autophagy. Functional analyses of transcription factors and cofactors identified C/EBPβ as a potent activator of autophagy. C/EBPβ is rhythmically expressed in the liver and is regulated by both circadian and nutritional signals. In cultured primary hepatocytes, C/EBPβ stimulates the program of autophagy gene expression and is sufficient to activate autophagic protein degradation. Adenoviral-mediated RNAi knockdown of C/EBPβ in vivo abolishes diurnal autophagy rhythm in the liver. Further, circadian regulation of C/EBPβ and autophagy is disrupted in mice lacking a functional liver clock. We have thus identified C/EBPβ as a key factor that links autophagy to biological clock and maintains nutrient homeostasis throughout light/dark cycles.

Biogenesis and Cargo Selectivity of Autophagosomes

Autophagy is a major catabolic pathway in eukaryotes, which is required for the lysosomal/vacuolar degradation of cytoplasmic proteins and organelles. Interest in the autophagy pathway has recently gained momentum largely owing to identification of multiple autophagy-related genes and recognition of its involvement in various physiological conditions. Here we review current knowledge of the molecular mechanisms regulating autophagy in mammals and yeast, specifically the biogenesis of autophagosomes and the selectivity of their cargo recruitment. We discuss the different steps of autophagy, from the signal transduction events that regulate it to the completion of this pathway by fusion with the lysosome/vacuole. We also review research on the origin of the autophagic membrane, the molecular mechanism of autophagosome formation, and the roles of two ubiquitin-like protein families and other structural elements that are essential for this process. Finally, we discuss the various modes of autophagy and highlight their functional relevance for selective degradation of specific cargos.

Abstract 3836: Identification of PTPsigma as an autophagic phosphatase

Abstract Macroautophagy is a dynamic process whereby portions of the cytosol are encapsulated in double-membrane vesicles and delivered to the lysosome for degradation. Phosphatidylinositol-3-phosphate (PI(3)P) is concentrated on autophagic vesicles and recruits effector proteins critical for this process. The production of PI(3)P by the class III phosphatidylinositol 3-kinase (PI3K), Vps34, has been well established; however, protein phosphatases which antagonize this early step in autophagy remain to be identified. To identify such enzymes, we screened human phosphatase genes by RNA interference (RNAi) and found that loss of PTPsigma, a dual-domain protein tyrosine phosphatase (PTP), increases cellular PI(3)P. The abundant PI(3)P-positive vesicles conferred by PTPsigma loss strikingly phenocopied those observed in amino acid-starved cells. Accordingly, we discovered that loss of PTPsigma hyperactivates both constitutive and induced autophagy. Finally, we found that PTPsigma localizes to PI(3)P-positive membranes in cells and this vesicular localization is enhanced during autophagy. Our findings propose a novel role for PTPsigma and provide insight into the regulation of autophagy. Mechanistic knowledge of this process is critical for understanding and targeting therapies for several human diseases, including cancer and Alzheimer's disease, in which abnormal autophagy may be pathological. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3836. doi:10.1158/1538-7445.AM2011-3836

Abstract 3772: The amplified cancer gene LAPTM4B plays a critical role in autophagosome maturation and promotes cancer cell survival during metabolic stress

Abstract Research has shown that lysosome-mediated autophagy is up regulated in response to various stimuli such as hypoxia, nutrient limitation, and drug-induced cytotoxic stress. In cancer progression, autophagy plays a cytoprotective role by generating needed nutrients or by eliminating damaged cellular components. Although autophagy has been recognized as a critical tumor cell survival mechanism and much is known about the induction and early steps of autophagy, the molecule(s) which control the later stages of autophagic progression are less clear. Previously, we have identified lysosomal-associated protein transmembrane 4b (LAPTM4B) as an amplified and over-expressed gene in breast cancer that is associated with de novo resistance to anthracycline chemotherapy and increased risk of distant metastatic recurrence. We reported that LAPTM4B overexpression alters intracellular trafficking of anthracyclines by retaining the drug in the cytoplasmic compartment thereby limiting drug-induced DNA damage in the nucleus. However, how this protein contributes to tumor cell survival in the setting of other types of stress requires further investigation. In the present studies, we used fluorescence microscopy to confirm LAPTM4B protein distribution in the lysosomal compartment of cells. Depletion of this gene results in increased lysosome membrane permeability as demonstrated by release from lysosomes of low to intermediate molecular weight dextran. Furthermore, we find that over expression of LAPTM4B renders cells resistant to apoptosis when placed in nutrient-free media suggesting a possible role of LAPTM4B in promoting lysosome-mediated autophagy. We induced autophagy by serum starvation in BT549 breast cancer cells with or without depletion of LAPTM4B. Autophagic progression was monitored by immunofluorescence for the autophagosome marker, LC3, and for degradation of P62 as evidence of autolysosome maturation. We find that depletion of LAPTM4B interrupted autophagosome maturation to autolysosomes as indicated by appearance of LC3 but absence of P62 degradation. In addition, we observed aggregation of autophagosomes in LAPTM4B-depleted cells and caspase activation, indicative of apoptosis. These events are a recognized sequela of blocked autophagic maturation. In summary, we find that ablation of LAPTM4B results in increased lysosome membrane permeability, inhibition of autophagosome-lysosome fusion, and toxic accumulation of autophagasomes triggering cell death. These results suggest a novel function for the amplified cancer gene, LAPTM4B, in promoting the later stages of autophagic maturation and tumor resistance to apoptosis-inducing stress. Targeting LAPTM4B may provide a new strategy to prevent metabolic adaptation of cancer cells by abolishing the cytoprotective effects of autophagy and increasing drug therapy efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3772. doi:10.1158/1538-7445.AM2011-3772

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-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-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-RasV12. 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-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-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.

Autophagy: Regulation by Energy Sensing

Autophagy is inhibited by the mTOR signaling pathway, which is stimulated by increased amino acid levels. When cellular energy production is compromised, AMP-activated protein kinase is activated, mTOR is inhibited and autophagy is stimulated. Two recent studies have shed light on the molecular mechanism by which AMPK controls autophagic flux.

Identification of PTPσ as an autophagic phosphatase

Macroautophagy is a dynamic process whereby portions of the cytosol are encapsulated in double-membrane vesicles and delivered to the lysosome for degradation. Phosphatidylinositol-3-phosphate (PtdIns3P) is concentrated on autophagic vesicles and recruits effector proteins that are crucial for this process. The production of PtdIns3P by the class III phosphatidylinositol 3-kinase Vps34, has been well established; however, protein phosphatases that antagonize this early step in autophagy remain to be identified. To identify such enzymes, we screened human phosphatase genes by RNA interference and found that loss of PTPσ, a dual-domain protein tyrosine phosphatase (PTP), increases levels of cellular PtdIns3P. The abundant PtdIns3P-positive vesicles conferred by loss of PTPσ strikingly phenocopied those observed in cells starved of amino acids. Accordingly, we discovered that loss of PTPσ hyperactivates both constitutive and induced autophagy. Finally, we found that PTPσ localizes to PtdIns3P-positive membranes in cells, and this vesicular localization is enhanced during autophagy. We therefore describe a novel role for PTPσ and provide insight into the regulation of autophagy. Mechanistic knowledge of this process is crucial for understanding and targeting therapies for several human diseases, including cancer and Alzheimer's disease, in which abnormal autophagy might be pathological.

Insufficient Activation of Autophagy Allows Cellular Damage to Accumulate in Critically Ill Patients

Abstract Context: Responses to critical illness, such as excessive inflammation and hyperglycemia, may trigger detrimental chain reactions that damage cellular proteins and organelles. Such responses to illness contribute to the risk of (nonresolving) multiple organ dysfunction and adverse outcome. Objective: We studied autophagy as a bulk degradation pathway able to remove toxic protein aggregates and damaged organelles and how these are affected by preventing hyperglycemia with insulin during critical illness. Design and Setting: Patients participated in a randomized study, conducted at a university hospital surgical/medical intensive care unit. Patients: We studied adult prolonged critically ill patients vs. controls. Interventions: Tolerating excessive hyperglycemia was compared with intensive insulin therapy targeting normoglycemia. Main Outcome Measures: We quantified (ultra)structural abnormalities and hepatic and skeletal muscle protein levels of key players in autophagy. Results: Morphologically, both liver and muscle revealed an autophagy-deficiency phenotype. Proteins involved in initiation and elongation steps of autophagy were induced 1.3- to 6.5-fold by critical illness (P < 0.01), but mature autophagic vacuole formation was 62% impaired (P = 0.05) and proteins normally degraded by autophagy accumulated up to 97-fold (P < 0.03). Mitophagy markers were unaltered or down-regulated (P = 0.05). Although insulin preserved hepatocytic mitochondrial integrity (P = 0.05), it further reduced the number of autophagic vacuoles by 80% (P = 0.05). Conclusions: Insufficient autophagy in prolonged critical illness may cause inadequate removal of damaged proteins and mitochondria. Such incomplete clearance of cellular damage, inflicted by illness and aggravated by hyperglycemia, could explain lack of recovery from organ failure in prolonged critically ill patients. These data open perspectives for therapies that activate autophagy during critical illness.

Mitochondrial fusion is an early and protective step of autophagy

Mitochondrial fusion is an early and protective step of autophagy

Abstract 4844: Dual effect of Akt on autophagy in pancreatic cancer cells

Abstract Background & Aims: Key steps in autophagic protein degradation are formation of autophagosomes (induction) and their fusion with lysosomes (progression). Akt is a major anti-apoptotic factor in cancer cells; however, its role in autophagy in general, and in pancreatic cancer (PaCa) in particular, is poorly understood. We recently showed that phosphatases PHLPP-I and -II are key negative regulators of Akt in PaCa cells. The present study aimed to elucidate the effects of Akt and PHLPPs on individual steps of autophagy and the role of autophagy in the prosurvival function of Akt in PaCa cells. Methods: We measured apoptosis by DNA fragmentation and caspase-3 activity; autophagy, by conversion of cytosolic LC3-I to autophagic LC3-II (with immunoblot) and LC3 immunofluorescence; autophagy induction, in cells pretreated with NH4Cl to block autophagy progression; autophagosomal fusion with lysosomes, by co-localization of the lysosomal marker LAMP-1 with LC3-II; and efficiency of protein degradation, by level of p62 protein, a specific autophagy target. Results: Akt inactivation by AktVIII inhibitor or siRNA, or by overexpression of PHLPP-I and -II all stimulated autophagic vacuole formation in MiaPaCa-2 and PANC-1 cells. Akt inactivation greatly facilitated LC3-I/LC3-II conversion both in the presence and absence of NH4Cl, indicating increased autophagy induction. At the same time, Akt inactivation inhibited autophagosomal fusion with lysosomes and efficiency of protein degradation, indicating decreased autophagy progression. Thus, Akt has dual effect on autophagy in PaCa cells: it inhibits autophagy induction but promotes autophagosomal fusion with lysosomes and hence autophagy progression. The overall effect of Akt is decreased accumulation of autophagic vacuoles, i.e., inhibition of autophagic death. To study the role of autophagy in apoptosis, we used 3-methyladenine or beclin siRNA to inhibit autophagy induction, and bafilomycin or chloroquine to block the fusion of autophagosomes with lysosomes. All these treatments stimulated apoptosis in PaCa cells, indicating the anti-apoptotic role of autophagy. In cells with inactivated Akt, blocking autophagy induction (e.g., with 3-methyladenine) further greatly stimulated apoptosis. This indicates that the ability of Akt to inhibit autophagy induction greatly diminishes its anti-apoptotic function. Differently, blocking autophagosomal fusion with lysosomes had little effect on apoptosis in cells in which Akt was inactivated, likely because in these cells autophagy progression was already inhibited. Conclusions: Akt inhibition hampers autophagy progression, thus promoting vacuolization and autophagic cell death. At the same time, Akt inhibition greatly stimulates autophagy induction, thus counteracting apoptosis. Therefore, to maximally stimulate apoptosis in PaCa cells we propose to apply Akt inhibitors in combination with inhibitors of autophagy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4844.