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Autophagy is a catabolic process that was described to play a critical role in advanced stages of cancer, wherein it maintains tumor cell homeostasis and growth by supplying nutrients. Autophagy is also described to support alternative cellular trafficking pathways, providing a non-canonical autophagy-dependent inflammatory cytokine secretion mechanism. Therefore, autophagy inhibitors have high potential in the treatment of cancer and acute inflammation. In our study, we identified compound 1 as an inhibitor of the ATG12-ATG3 protein-protein interaction. We focused on the systematic modification of the original hit 1, a casein kinase 2 (CK2) inhibitor, to find potent disruptors of ATG12-ATG3 protein-protein interaction. A systematic modification of the hit structure led us to a wide plethora of compounds that maintain its ATG12-ATG3 inhibitory activity, which could act as a viable starting point to design new compounds with diverse therapeutic applications., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Adi Kimchi reports financial support was provided by YEDA Technology Transfer. Radim Nencka reports financial support was provided by Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences. Adi Kimchi has patent #WO2023126951 issued to YEDA Technology Transfer; IOCB CAS. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Autophagy related 4B (ATG4B) which regulates autophagy by promoting the formation of autophagosome through reversible modification of LC3, is closely related to cancer cell growth and drug resistance, and therefore is an attractive therapeutic target. Recently, ATG4B inhibitors have been reported, yet with drawbacks including weak potency. To discover more promising ATG4B inhibitors, we developed a high-throughput screening (HTS) assay and identified a new ATG4B inhibitor named DC-ATG4in. DC-ATG4in directly binds to ATG4B and inhibits its enzyme activity with an IC 50 of 3.08 ± 0.47 μM. We further confirmed that DC-ATG4in is an autophagy inhibitor and blocks autophagy induced by Sorafenib in Hepatocellular Carcinoma (HCC) cells. More importantly, combination of DC-ATG4in with Sorafenib synergized the cancer cell killing effect and proliferation inhibition activities on HCC cells. Our data suggested that inactivation of autophagy via ATG4B inhibition may be a viable strategy to sensitize existing targeted therapy such as Sorafenib in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

One possible strategy for modulating autophagy is to disrupt the critical protein-protein interactions (PPIs) formed during this process. Our attention is on the autophagy-related 12 (ATG12)-autophagy-related 5 (ATG5)-autophagy-related 16-like 1 (ATG16L1) heterotrimer complex, which is responsible for ATG8 translocation from ATG3 to phosphatidylethanolamine. In this work, we discovered a compound with an ( E )-3-(2-furanylmethylene)-2-pyrrolidinone core moiety ( T1742 ) that blocked the ATG5-ATG16L1 and ATG5-TECAIR interactions in the in vitro binding assay (IC 50 = 1-2 μM) and also exhibited autophagy inhibition in cellular assays. The possible binding mode of T1742 to ATG5 was predicted through molecular modeling, and a batch of derivatives sharing essentially the same core moiety were synthesized and tested. The outcomes of the in vitro binding assay and the flow cytometry assay of those newly synthesized compounds were generally consistent. This work has validated our central hypothesis that small-molecule inhibitors of the PPIs involving ATG5 can tune down autophagy effectively, and their pharmaceutical potential may be further explored.

Autophagy is a non-selective action in which cells degrade parts of themselves, reusing degraded cellular components. Among autophagy-related gene (ATG) family members, ATG4 proteins play crucial roles in the microtubule-associated protein 1 light chain 3 (LC3) phosphatidylethanolamine (PE) system which is essential for autophagosome maturation. Although autophagy has been shown to be involved in osteoclastic bone resorption, the role of ATG4/LC3 in bone resorption remains unclear. When mouse bone marrow cells were treated with various concentrations of NSC185058 (NSC), a specific inhibitor of ATG4B, 1 h prior to treatment with receptor activator of NF-κB ligand (RANKL) in the presence of macrophage colony stimulating factor (M-CSF), NSC inhibited osteoclastogenesis in a dose-dependent manner. Addition of NSC in the late stages of osteoclast differentiation suppressed multinucleation and reduced the expression of markers for mature osteoclasts such as Dc-stamp, Mmp9, and Ctsk. NSC also suppressed actin ring formation and pit formation in mature osteoclasts. When a periodontitis model involving eight-week-old male mice in which the right maxillary second molar had been ligated with silk thread was injected with or without NSC, alveolar bone resorption was suppressed by a decrease in the number of osteoclasts in the NSC-treated group. These results suggest that LC3 is important for the maturation of osteoclasts and that LC3 inhibition is a new therapeutic strategy for periodontal disease., Competing Interests: Declaration of competing interest The authors have declared no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Advanced gastric cancer (GCa) remains highly lethal due to the lack of effective therapies. Identifying promising therapeutic targets and developing effective treatment against GCa are urgently needed. Through mRNA and protein analysis of GCa clinical tumor samples, we found that autophagy-related gene 4B (ATG4B) was overexpressed in GCa tumors and that its high expression was associated with patients' poor prognosis. Knockdown of ATG4B significantly inhibited GCa cell survival and tumor growth. To further probe the role of ATG4B in GCa by pharmacological means, we screened an in-house marine natural compound library against ATG4B and identified Azalomycin F4a (Am-F4a) as a novel and potent ATG4B inhibitor. Am-F4a directly bound to ATG4B with high affinity and effectively suppressed GCa cell autophagy via inhibition of ATG4B both in vitro and in vivo. Moreover, Am-F4a or ATG4B knockdown significantly suppressed tumor growth as well as GCa cell migration and invasion. Am-F4a effectively blocked the metastatic progression of primary GCa and sensitized tumors to chemotherapy. Taken together, our findings indicate that ATG4B is a potential therapeutic target against GCa and the natural product Am-F4a is a novel ATG4B inhibitor that can be further developed for the treatment of GCa., (© 2022. The Author(s).)

Background & Aims: Autophagy-related gene 3 (ATG3) is an enzyme mainly known for its actions in the LC3 lipidation process, which is essential for autophagy. Whether ATG3 plays a role in lipid metabolism or contributes to non-alcoholic fatty liver disease (NAFLD) remains unknown., Methods: By performing proteomic analysis on livers from mice with genetic manipulation of hepatic p63, a regulator of fatty acid metabolism, we identified ATG3 as a new target downstream of p63. ATG3 was evaluated in liver samples from patients with NAFLD. Further, genetic manipulation of ATG3 was performed in human hepatocyte cell lines, primary hepatocytes and in the livers of mice., Results: ATG3 expression is induced in the liver of animal models and patients with NAFLD (both steatosis and non-alcoholic steatohepatitis) compared with those without liver disease. Moreover, genetic knockdown of ATG3 in mice and human hepatocytes ameliorates p63- and diet-induced steatosis, while its overexpression increases the lipid load in hepatocytes. The inhibition of hepatic ATG3 improves fatty acid metabolism by reducing c-Jun N-terminal protein kinase 1 (JNK1), which increases sirtuin 1 (SIRT1), carnitine palmitoyltransferase 1a (CPT1a), and mitochondrial function. Hepatic knockdown of SIRT1 and CPT1a blunts the effects of ATG3 on mitochondrial activity. Unexpectedly, these effects are independent of an autophagic action., Conclusions: Collectively, these findings indicate that ATG3 is a novel protein implicated in the development of steatosis., Lay Summary: We show that autophagy-related gene 3 (ATG3) contributes to the progression of non-alcoholic fatty liver disease in humans and mice. Hepatic knockdown of ATG3 ameliorates the development of NAFLD by stimulating mitochondrial function. Thus, ATG3 is an important factor implicated in steatosis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest related to the study. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cycloastragenol (CAG), a secondary metabolite from the roots of Astragalus zahlbruckneri , has been reported to exert anti‑inflammatory effects in heart, skin and liver diseases. However, its role in asthma remains unclear. The present study aimed to investigate the effect of CAG on airway inflammation in an ovalbumin (OVA)‑induced mouse asthma model. The current study evaluated the lung function and levels of inflammation and autophagy via measurement of airway hyperresponsiveness (AHR), lung histology examination, inflammatory cytokine measurement and western blotting, amongst other techniques. The results demonstrated that CAG attenuated OVA‑induced AHR in vivo . In addition, the total number of leukocytes and eosinophils, as well as the secretion of inflammatory cytokines, including interleukin (IL)‑5, IL‑13 and immunoglobulin E were diminished in bronchoalveolar lavage fluid of the OVA‑induced murine asthma model. Histological analysis revealed that CAG suppressed inflammatory cell infiltration and goblet cell secretion. Notably, based on molecular docking simulation, CAG was demonstrated to bind to the active site of autophagy‑related gene 4‑microtubule‑associated proteins light chain 3 complex, which explains the reduced autophagic flux in asthma caused by CAG. The expression levels of proteins associated with autophagy pathways were inhibited following treatment with CAG. Taken together, the results of the present study suggest that CAG exerts an anti‑inflammatory effect in asthma, and its role may be associated with the inhibition of autophagy in lung cells.

Breast cancer (BRCA) is one of the most threatening cancer types, especially among the female population. 70% of breast cancer are estrogen receptor α (ERα) positive and endocrine therapy is effective to decrease breast cancer risk. Autophagy, a highly conserved cellular recycling process, has been regarded to serve a protective role in BRCA. Autophagy-related gene 13 (Atg13) is participated in autophagy and is critical to autophagy initiation. Briefly, we observed that ERα, a well-known transcription factor that can promote breast cancer cell proliferation, expressed higher in breast cancer tissues. Moreover, ERα had a significant positive correlation with Atg13 and may be able to regulate the transcription of Atg13 via binding the promoter region of Atg13. Surprisingly, Oxabicycloheptene sulfonate (OBHS), the drug that we reported as a selective estrogen receptor modulator (SERM) before, may have the ability to decrease the expression of ERα and suppress the autophagy. In conclusion. We found that ERα could be involved in autophagy by binding the promoter of Atg13, and compound OBHS may be able to affect the viability of breast cancer cells by decreasing the expression of ERα and Atg13., Competing Interests: Declaration of competing interest I would like to declare that the work described was original that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed. No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A collection of 9050 natural products, their derivatives, and mimetics, was virtually screened against the human Atg3-Atg8 (Atg - autophagy) binding scaffold. By blocking this interaction, the lipidation of Atg8 does not occur and the formation of autophagosomes is inhibited. Forty-three (43) potential ligands were tested using enhanced Green Fluorescent Protein (eGFP) tagged LC3, the human ortholog of Atg8, in MCF7 breast cancer cells. Three hits showed single digit µM IC 50 values with AT110, an isoflavone derivative, being the best at 1.2 ± 0.6 µM. Molecular modelling against Atg8 in conjunction with structural activity relationship (SAR) strongly supports the binding to this target. Testing in a panel of cancer cell lines showed little cytotoxic effect as compared to chloroquine. However, same concentration of AT110 was shown to be toxic to young zebrafish embryos. This can be explained in terms of the autophagy process being very active in the zebrafish embryos rendering them susceptible to AT110 whereas in the cancer cells tested the autophagy is not usually active. Nevertheless, AT110 blocks autophagy flux in the zebrafish confirming that the ligand is modulating autophagy. A small molecule non-cytotoxic autophagy inhibitor would open the door for adjunct therapies to bolster many established anticancer drugs, reducing their efficacious concentration thus limiting undesirable site effects. In addition, since many cancer types rely on the autophagy mechanism to survive a therapeutic regime, recurrence can potentially be reduced. The discovery of AT110 is an important step in establishing such an adjunct therapy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The novel betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused the coronavirus disease 19 (COVID-19) pandemic due to its high transmissibility and early immunosuppression. Previous studies on other betacoronaviruses suggested that betacoronavirus infection is associated with the host autophagy pathway. However, it is unclear whether any components of autophagy or virophagy can be therapeutic targets for COVID-19 treatment. In this report, we examined the antiviral effect of four well-characterized small molecule inhibitors that target the key cellular factors involved in key steps of the autophagy pathway. They include small molecules targeting the ULK1/Atg1 complex involved in the induction stage of autophagy (ULK1 inhibitor SBI0206965), the ATG14/Beclin1/VPS34 complex involved in the nucleation step of autophagy (class III PI3-kinase inhibitor VPS34-IN1), and a widely-used autophagy inhibitor that persistently inhibits class I and temporary inhibits class III PI3-kinase (3-MA) and a clinically approved autophagy inhibitor that suppresses autophagy by inhibiting lysosomal acidification and prevents the formation of autophagolysosome (HCQ). Surprisingly, not all the tested autophagy inhibitors suppressed SARS-CoV-2 infection. We showed that inhibition of class III PI3-kinase involved in the initiation step of both canonical and noncanonical autophagy potently suppressed SARS-CoV-2 at a nano-molar level. In addition, this specific kinase inhibitor VPS34-IN1, and its bioavailable analogue VVPS34-IN1, potently inhibited SARS-CoV-2 infection in ex vivo human lung tissues. Taken together, class III PI3-kinase may be a possible target for COVID-19 therapeutic development., (© 2020 Wiley Periodicals LLC.)

Numerous microRNAs participate in regulating the pathological process of atherosclerosis. We have found miR-130a is one of the most significantly down-regulated microRNAs in arteriosclerosis obliterans. Our research explored the function of miR-130a in regulating proliferation by controlling autophagy in arteriosclerosis obliterans development. A Gene Ontology (GO) enrichment analysis of miR-130a target genes indicated a correlation between miR-130a and cell proliferation. Thus, cell cycle, CCK-8 assays and Western blot analysis were performed, and the results indicated that miR-130a overexpression in vascular smooth muscle cells (VSMCs) significantly attenuated cell proliferation, which was validated by an in vivo assay in a rat model. Moreover, autophagy is thought to be involved in the regulation of proliferation. As our results indicated, miR-130a could inhibit autophagy, and ATG2B was predicted to be a target of miR-130a. The autophagy inhibition effect of miR-130a overexpression was consistent with the effect of ATG2B knockdown. The results that ATG2B plasmids and miR-130a mimics were cotransfected in VSMCs further confirmed our conclusion. In addition, by using immunohistochemistry, the positive results of LC3 II/I and ATG2B in the rat model and artery vascular tissues from the patient were in accordance with in vitro data. In conclusion, our data demonstrate that miR-130a inhibits VSMCs proliferation via ATG2B, which indicates that miR-130a could be a potential therapeutic target that regulates autophagy in atherosclerosis obliterans., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Acanthopanax senticosus (Rupr. et Maxim) Harms (ASH), also known as Siberian ginseng or eleuthero, is a hardy shrub native to China, Korea, Russia and the northern region of Japan. ASH is used for the treatment of several diseases such as heart disease, hypertension, rheumatoid arthritis, allergies, chronic bronchitis, diabetes and cancer. In the present study, the inhibitory effect of the root extract of ASH (ASHE) on HuH‑7 and HepG2 liver cancer cells was examined. ASHE suppressed liver cancer cell proliferation by inducing cell cycle arrest at the G0/G1 phase, as well as apoptosis, as indicated by the increased number of Annexin V and 7‑AAD‑positive cells. Furthermore, the expression of LC3‑II, an autophagy marker, in these cells also increased post treatment with ASHE. LC3‑II induction was further enhanced by co‑treatment with chloroquine. Fluorescence and transmission electron micrographs of ASHE‑treated liver cancer cells showed the presence of an increased number of autophagic vesicles. A decreased protein expression level of run domain Beclin‑1‑interacting and cysteine‑rich domain‑containing, an autophagy inhibitor, with no change in RUBCN mRNA expression was observed, indicating activation of the autophagosome‑lysosome fusion step of autophagy. In conclusion, ASHE exerts cytostatic activity on liver cancer cells via both apoptosis and autophagy, and may serve as a potential therapeutic agent for management of liver cancer and autophagy‑related diseases.

Atg4, a pivotal macroautophagy/autophagy-related cysteine protein family, which regulate autophagy through either cleaving Atg8 homologs for its further lipidation or delipidating Atg8 homologs from the autophagosome. There are four homologs, Atg4A, Atg4B, Atg4C, and Atg4D. Among them, an increasing amount of evidence indicates that Atg4B possessed superior catalytic efficiency toward the Atg8 substrate, as well as regulates autophagy process and plays a key role in the development of several human cancers. Recently, efforts have been contributed to the exploration of Atg4B inhibitors or activators. In this review, we comprehensively clarify the function of Atg4B in autophagy and cancer biology, as well as the relationship between pharmacological function and structure-activity of small molecule drugs targeting Atg4B. The development of novel drugs targeting Atg4B could be well applied in the clinical practice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Autophagy is an evolutionarily conserved self-cannibalization process commonly found in all eukaryotic cells. Through autophagy, long-lived or damaged organelles, superfluous proteins, and pathogens are sequestered and encapsulated into the double-membrane autophagosomes prior to fusion with lysosomes for ultimate degradation and recycling. Given that autophagy is deemed both protective and detrimental in malignancies, the clinical therapeutic utilization of autophagy modulators in cancer has attracted immense attentions over the past decades. Dependence of tumor cells on autophagy during amino acid insufficiency or deprivation has prompted us to explore the underlying autophagy regulatory mechanisms to inject amino acid degrading enzymes and enzyme-based strategies into therapeutic maneuvers of autophagy in cancer., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The SARS-CoV-2 pandemic necessitates a review of the molecular mechanisms underlying cellular infection by coronaviruses, in order to identify potential therapeutic targets against the associated new disease (COVID-19). Previous studies on its counterparts prove a complex and concomitant interaction between coronaviruses and autophagy. The precise manipulation of this pathway allows these viruses to exploit the autophagy molecular machinery while avoiding its protective apoptotic drift and cellular innate immune responses. In turn, the maneuverability margins of such hijacking appear to be so narrow that the modulation of the autophagy, regardless of whether using inducers or inhibitors (many of which are FDA-approved for the treatment of other diseases), is usually detrimental to viral replication, including SARS-CoV-2. Recent discoveries indicate that these interactions stretch into the still poorly explored noncanonical autophagy pathway, which might play a substantial role in coronavirus replication. Still, some potential therapeutic targets within this pathway, such as RAB9 and its interacting proteins, look promising considering current knowledge. Thus, the combinatory treatment of COVID-19 with drugs affecting both canonical and noncanonical autophagy pathways may be a turning point in the fight against this and other viral infections, which may also imply beneficial prospects of long-term protection.

Osteosarcoma (OS) is a kind of primary bone cancer that is considered as the leading cause of children death. Surgery and chemotherapy are considered as common treatment approaches for OS; the rate of survival for patients is almost 60-70%. Besides the used therapeutic approaches, it seems that there is a crucial need to launch new treatments for OS. In this regard, more understanding about cellular and molecular pathways involved in OS can contribute to recovery and develop new therapeutic platforms. Autophagy is a cellular machinery that digests and degrades dysfunctional proteins and organelles, so it can regulate the cell proliferation and survival. Most of the time, OS cells use autophagy to increase their survival and proliferation and to gain the ability to resist chemotherapy. Although, there are several controversial evidences on how OS cells use autophagy. A variety of cellular and molecular pathways, that is, microRNAs (miRNAs) can modulate autophagy. MiRNAs are some endogenous, approximately 22 nucleotide RNAs that have an important role in posttranscriptional regulation of mRNAs by targeting them. There are many evidences that the various miRNA expressions in OS cells are dysregulated, so it can propel a normal cell to cancerous one by influencing the cell survival, apoptosis, and autophagy, and eventually increased chemoresitance. Hence, miRNAs can be considered as new biomarkers for OS diagnosis, and according to the role of autophagy in OS progression, miRNAs can use inhibiting or promoting autophagy agents. The present review summarizes the effects of aberrant expression of miRNAs in OS diagnosis and treatment with focus on their roles in autophagy., (© 2020 International Union of Biochemistry and Molecular Biology.)

Autophagy, a catabolic recycling process, has been implicated as a critical pathway in cancer. Its role in maintaining cellular homeostasis helps to nourish hypoxic, nutrient-starved tumors and protects them from chemotherapy-induced death. Recent efforts to target autophagy in cancer have focused on kinase inhibition, which has led to molecules that lack specificity due to the multiple roles of key kinases in this pathway. For example, the lipid kinase VPS34 is present in two multiprotein complexes responsible for the generation of phosphatidylinositol-3-phosphate. Complex I generates the autophagosome, and Complex II is crucial for endosomal trafficking. Molecules targeting VPS34 inhibit both complexes, which inhibits autophagy but causes undesirable defects in vesicle trafficking. The lack of specific autophagy modulators has limited the utility of autophagy inhibition as a therapeutic strategy. We hypothesize that disruption of the Beclin 1-ATG14L protein-protein interaction, which is required for the formation, proper localization, and function of VPS34 Complex I but not Complex II, will disrupt Complex I formation and selectively inhibit autophagy. To this end, a high-throughput, cellular NanoBRET assay was developed targeting this interaction. An initial screen of 2560 molecules yielded 19 hits that effectively disrupted the interaction, and it was confirmed that one hit disrupted VPS34 Complex I formation and inhibited autophagy. In addition, the molecule did not disrupt the Beclin 1-UVRAG interaction, critical for VPS34 Complex II, and thus had little impact on vesicle trafficking. This molecule is a promising new tool that is critical for understanding how modulation of the Beclin 1-ATG14L interaction affects autophagy. More broadly, its discovery demonstrates that targeting protein-protein interactions found within the autophagy pathway is a viable strategy for the discovery of autophagy-specific probes and therapeutics.

Background: Neoantigens are newly formed antigens that have not been previously recognized by the immune system. They may arise from altered tumor proteins that form as a result of mutations. Although neoantigens have recently been linked to antitumor immunity in long-term survivors of cancers, such as melanoma and colorectal cancer, their prognostic and immune-modulatory role in many cancer types remains undefined., Objective: The purpose of this study is to identify prognostic markers for long-term extrahepatic cholangiocarcinoma (EHCC) survival., Methods: We investigated neoantigens in EHCC, a rare, aggressive cancer with a 5-year overall survival rate lower than 10%, using a combination of whole-exome sequencing (WES), RNA sequencing (RNA-seq), computational biophysics, and immunohistochemistry., Results: Our analysis revealed a decreased neutrophil infiltration-related trend of high-quality neoantigen load with IC50 <500 nM (r=-0.445, P=0.043). Among 24 EHCC patients examined, we identified four long-term survivors with WDFY3 neoantigens and none with WDFY3 neoantigens in the short-term survivors. The WDFY3 neoantigens are associated with a lower infiltration of neutrophils (p=0.013), lower expression of CCL5 (p=0.025), CXCL9 (p=0.036) and TIGIT (p=0.016), and less favorable prognosis (p=0.030). In contrast, the prognosis was not significantly associated with tumor mutation burden, neoantigen load, or immune cell infiltration., Conclusion: We suggest that the WDFY3 neoantigens may affect prognosis by regulating antitumor immunity and that the WDFY3 neoantigens may be harnessed as potential targets for immunotherapy of EHCC., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The cellular recycling process of macroautophagy, which is the mechanism by which cellular material is delivered to lysosomes via double membraned vesicles called autophagosomes, is intimately connected to programmed cell death pathways, especially apoptosis. In this article, I discuss some underlying mechanisms and their implications for improving cancer therapy and propose that the approaches that have been taken to understand the autophagy-apoptosis connection to enhance cancer drug action can serve as a model for the kinds of information that should be developed to understand how autophagy controls other biological processes as well., (© 2020 Elsevier Inc. All rights reserved.)

Targeting autophagy is a promising therapeutic strategy for cancer treatment. As a result, the identification of novel autophagy inhibitors is an emerging field of research. Herein, we report the development of a novel AlphaScreen HTS assay that combined with a MS-based assay and a structure-based high-throughput virtual screening have enabled the identification of benzo[cd]indol-2(1H)-one as a novel scaffold that targets Atg4B. Thus, an initial screening campaign led to the identification of NSC126353 and NSC611216 bearing a chlorohydrin moiety. Structural-activity relationship analysis of the initial hits provided an optimized lead, compound 33, bearing a 7-aminobenzo[cd]indol-2-[1H]-one scaffold and a propyl group replacing the chlorine. Inhibition of autophagy was also investigated in cells by measuring LC3-II and p62 protein levels. Moreover, the synergistic effect of 33 combined with oxaliplatin resulted in an enhanced cell death in the human colorectal adenocarcinoma cell line HT-29. We are convinced that the developed AlphaScreen and MS-based assays can be key tools enabling the high-throughput identification of novel Atg4B inhibitors. Moreover, the aminobenzo[cd]indol-2-[1H]-one scaffold represents a novel chemotype for the further development of small molecule inhibitors of Atg4B., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Combination treatment is a promising strategy to improve prognosis of hepatocellular carcinoma (HCC). Sorafenib is a traditional first-line agent approved for the treatment of advanced HCC, though with limited efficacy. Previously, we reported that lonafarnib, an orally bioavailable non-peptide inhibitor targeting farnesyltransferase, synergizes with sorafenib against the growth of HCC cells. In the present study, we aim to clarify the underlying mechanism of this combination strategy. Initially, using in vitro HCC cell model, we confirmed that synergistic treatment of lonafarnib and sorafenib suppressed cell viability and colony formation, and induced cell death. We then found conversion of LC3-I to LC3-II via combination the treatment and observed formation of autophagosomes by electron microscopy. Knockdown of ATG3 inhibited the autophagic flux induced by the combination treatment. Furthermore, we demonstrated that drug-eliciting autophagy selectively promoted the degradation of cyclin D1 in a lysosome-dependent manner and subsequently inhibited DNA synthesis through downregulating the phosphorylation of Rb protein. In conclusion, our results provide a deeper insight into the mechanism for the combination treatment of lonafarnib and sorafenib in HCC therapy.

Macroautophagy/autophagy is a complex self-degradative mechanism responsible for clearance of non functional organelles and proteins. A range of factors influences the autophagic process, and disruptions in autophagy-related mechanisms lead to disease states, and further exacerbation of disease. Despite in-depth research into autophagy and its role in pathophysiological processes, the resources available to use it for therapeutic purposes are currently lacking. Herein we report the Autophagy Small Molecule Database (AutophagySMDB; http://www.autophagysmdb.org/ ) of small molecules and their cognate protein targets that modulate autophagy. Presently, AutophagySMDB enlists ~10,000 small molecules which regulate 71 target proteins. All entries are comprised of information such as EC50 (half maximal effective concentration), IC50 (half maximal inhibitory concentration), Kd (dissociation constant) and Ki (inhibition constant), IUPAC name, canonical SMILE, structure, molecular weight, QSAR (quantitative structure activity relationship) properties such as hydrogen donor and acceptor count, aromatic rings and XlogP. AutophagySMDB is an exhaustive, cross-platform, manually curated database, where either the cognate targets for small molecule or small molecules for a target can be searched. This database is provided with different search options including text search, advanced search and structure search. Various computational tools such as tree tool, cataloging tools, and clustering tools have also been implemented for advanced analysis. Data and the tools provided in this database helps to identify common or unique scaffolds for designing novel drugs or to improve the existing ones for autophagy small molecule therapeutics. The approach to multitarget drug discovery by identifying common scaffolds has been illustrated with experimental validation. Abbreviations: AMPK: AMP-activated protein kinase; ATG: autophagy related; AutophagySMDB: autophagy small molecule database; BCL2: BCL2, apoptosis regulator; BECN1: beclin 1; CAPN: calpain; MTOR: mechanistic target of rapamycin kinase; PPARG: peroxisome proliferator activated receptor gamma; SMILES: simplified molecular input line entry system; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription.

Schwann cells are the main supportive cells of the peripheral nerves. Schwann cells suffer inhibition of autophagy under hyperglycemia treatment in diabetic peripheral neuropathy (DPN). However, the exact mechanism is still not fully elucidated. We first observed the decrease of autophagy markers (LC3-II/LC3-I, P62) in the sciatic nerves of diabetic mice vs. normal mice, accompanied with the loss of myelinated nerve fibers and abnormal myelin sheath. In line with this, LC3-II/LC3-I and P62 were also significantly reduced in high glucose-treated rat Schwann cell 96 (RSC96) cells compared with normal glucose-treated cells. Furthermore, we found that trichostatin A [an inhibitor of histone deacetylase (HDAC)] evidently improved LC3-II/LC3-I in high glucose-treated RSC96 cells, without an effect on P62 expression. Again, HDAC1 and HDAC5 were revealed to be increased in RSC96 cells stimulated with high glucose. Inhibition of HDAC1 but not HDAC5 by small hairpin RNA vector enhanced LC3-II/LC3-I in high glucose-cultured RSC96 cells. In addition, LC3-II conversion regulators [autophagy-related protein (Atg)3, Atg5, and Atg7] were detected in high glucose-treated and HDAC1-knockdown RSC96 cells, and Atg3 was proven to be the key target of HDAC1. The presuppression of Atg3 offset the improvement of LC3-II/LC3-I resulting from HDAC1 inhibition in high glucose-treated RSC96 cells. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway was activated in RSC96 cells treated with high glucose, which was indicated by increased STAT3 phosphorylation. Blocking STAT3 phosphorylation by chemical inhibitor AG490 induced HDAC1 down-regulation followed by increases in Atg3 and LC3-II/LC3-I. Interestingly, we also found that AG490 treatment enhanced P62 expression in high glucose-stimulated RSC96 cells. Taken together, our findings demonstrate that hyperglycemia inhibits LC3-II/LC3-I in an HDAC1-Atg3-dependent manner and decreases P62 expression in an HDAC-independent manner via the JAK-STAT3 signaling pathway in the Schwann cells of DPN.-Du, W., Wang, N., Li, F. Jia, K., An, J., Liu, Y., Wang, Y., Zhu, L., Zhao, S. Hao, J. STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

During macroautophagic stress, autophagosomes can be produced continuously and in high numbers. Many different organelles have been reported as potential donor membranes for this sustained autophagosome growth, but specific machinery to support the delivery of lipid to the growing autophagosome membrane has remained unknown. Here we show that the autophagy protein, ATG2, without a clear function since its discovery over 20 yr ago, is in fact a lipid-transfer protein likely operating at the ER-autophagosome interface. ATG2A can bind tens of glycerophospholipids at once and transfers lipids robustly in vitro. An N-terminal fragment of ATG2A that supports lipid transfer in vitro is both necessary and fully sufficient to rescue blocked autophagosome biogenesis in ATG2A/ATG2B KO cells, implying that regulation of lipid homeostasis is the major autophagy-dependent activity of this protein and, by extension, that protein-mediated lipid transfer across contact sites is a principal contributor to autophagosome formation., (© 2019 Valverde et al.)

Autophagy is an evolutionarily conserved pathway to degrade damaged proteins and organelles for subsequent recycling in cells during times of nutrient deprivation. This process plays an important role in tumor development and progression, allowing cancer cells to survive in nutrient-poor environments. The plant kingdom provides a powerful source for new drug development to treat cancer. Several plant extracts induce autophagy in cancer cells. However, little is known about the role of plant extracts in autophagy inhibition, particularly autophagy-related (ATG) proteins. In this study, we employed S-tagged gamma-aminobutyric acid receptor associated protein like 2 (GABARAPL2) as a reporter to screen 48 plant extracts for their effects on the activity of autophagy protease ATG4B. Xanthium strumarium and Tribulus terrestris fruit extracts were validated as potential ATG4B inhibitors by another reporter substrate MAP1LC3B-PLA 2 . The inhibitory effects of the extracts on cellular ATG4B and autophagic flux were further confirmed. Moreover, the plant extracts significantly reduced colorectal cancer cell viability and sensitized cancer cells to starvation conditions. The fruit extract of X. strumarium consistently diminished cancer cell migration and invasion. Taken together, the results showed that the fruit of X. strumarium may have an active ingredient to inhibit ATG4B and suppress the proliferation and metastatic characteristics of colorectal cancer cells.

Autophagy is an intracellular degradation/recycling pathway that provides nutrients and building blocks to cellular metabolism and keeps the cytoplasm clear of obsolete proteins and organelles. During recent years, dysregulated autophagy activity has been reported to be a characteristic of many different disease types, including cancer and neurodegenerative disorders. This has created a strong case for development of autophagy modulating compounds as potential treatments for these diseases. Inhibitors of autophagy have been proposed as a therapeutic intervention in, e.g., advanced cancer, and inhibiting the cysteine protease Atg4B has been put forward as a main strategy to block autophagy. We recently identified and demonstrated -both in vitro and in vivo - that compounds with a benzotropolone basic structure targeting Atg4B, can significantly slow down tumor growth and potentiate the effect of classical chemotherapy. In this study we report the synthesis and inhibition profile of new benzotropolone derivatives with additional structural modifications at 6 different positions. To obtain a solid inhibition profile, all compounds were evaluated on three levels, including two cell-based assays to confirm autophagy and intracellular Atg4B inhibition and an SDS-PAGE-based experiment to assess in vitro Atg4B affinity. Several molecules with a promising profile were identified., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Macroautophagy/autophagy inhibition is a novel anticancer therapeutic strategy, especially for tumors driven by mutant RAS. Here, we demonstrate that autophagy inhibition in RAS-mutated cells induces epithelial-mesenchymal transition (EMT), which is associated with enhanced tumor invasion. This is at least partially achieved by triggering the NFKB/NF-κB pathway via SQSTM1/p62. Knockdown of ATG3 or ATG5 increases oncogenic RAS-induced expression of ZEB1 and SNAI2/Snail2, and activates NFKB activity. Depletion of SQSTM1 abolishes the activation of the NFKB pathway induced by autophagy inhibition in RAS-mutated cells. NFKB pathway inhibition by depletion of RELA/p65 blocks this EMT induction. Finally, accumulation of SQSTM1 protein correlates with loss of CDH1/E-cadherin expression in pancreatic adenocarcinoma. Together, we suggest that combining autophagy inhibition with NFKB inhibitors may therefore be necessary to treat RAS-mutated cancer. Abbreviations: 4-OHT: 4-hydroxytamoxifen; DIC: differential interference contrast; EMT: epithelial-mesenchymal transition; ESR: estrogen receptor; MAPK/ERK: mitogen-activated protein kinase; iBMK: immortalized baby mouse kidney epithelial cells; MET: mesenchymal-epithelial transition; PI3K: phosphoinositide 3-kinase; RNAi: RNA interference; TGFB/TGF-β: transforming growth factor beta; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6.

Macroautophagy is an evolutionarily conserved cellular maintenance program, meant to protect the brain from premature aging and neurodegeneration. How neuronal autophagy, usually loosing efficacy with age, intersects with neuronal processes mediating brain maintenance remains to be explored. Here, we show that impairing autophagy in the Drosophila learning center (mushroom body, MB) but not in other brain regions triggered changes normally restricted to aged brains: impaired associative olfactory memory as well as a brain-wide ultrastructural increase of presynaptic active zones (metaplasticity), a state non-compatible with memory formation. Mechanistically, decreasing autophagy within the MBs reduced expression of an NPY-family neuropeptide, and interfering with autocrine NPY signaling of the MBs provoked similar brain-wide metaplastic changes. Our results in an exemplary fashion show that autophagy-regulated signaling emanating from a higher brain integration center can execute high-level control over other brain regions to steer life-strategy decisions such as whether or not to form memories.

Transcription and DNA damage repair act in a coordinated manner. Recent studies have shown that double-strand DNA breaks (DSBs) are repaired in a transcription-coupled manner. Active transcription results in a faster recruitment of DSB repair factors and expedites DNA repair. On the other hand, transcription is repressed by DNA damage through multiple mechanisms. We previously reported that TLP, a TATA box-binding protein (TBP) family member that functions as a transcriptional regulator, is also involved in DNA damage-induced apoptosis. However, the mechanism by which TLP affects DNA damage response was largely unknown. Here we show that TLP-mediated global transcriptional repression after DSBs is crucial for apoptosis induction by DNA-damaging agents such as etoposide and doxorubicin. Compared to control cells, TLP-knockdown cells were resistant to etoposide-induced apoptosis and exhibited an elevated level of global transcription after etoposide exposure. DSBs were efficiently removed in transcriptionally hyperactive TLP-knockdown cells. However, forced transcriptional shutdown using transcriptional inhibitors α-amanitin and 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) slowed down DSB repair and resensitized TLP-knockdown cells to etoposide. Taken together, these results indicate that TLP is a critical determinant as to how cells respond to DSBs and triggers apoptosis to cells that have sustained DNA damage.

Human Atg4 homologs are cysteine proteases, which play key roles in the macroautophagy/autophagy process by cleaving Atg8 homologs for conjugation to lipid membranes and for deconjugation of Atg8 homologs from membranes. Expression of ATG4B is significantly increased in colorectal cancer cells compared to normal cells, suggesting that ATG4B may be important for cancer biology. Inhibition of ATG4B may reduce the autophagy activity, thereby sensitizing cancer cells to therapeutic agents. Thus, developing specific and potent ATG4B inhibitors for research as well as for potential therapeutic uses is highly needed. In this study, we integrated in silico screening and in vitro assays to discover a potent ATG4B inhibitor, named S130, from a noncommercial library. This chemical binds to ATG4B with strong affinity and specifically suppresses the activity of ATG4B but not other proteases. S130 did not cause the impairment of autophagosome fusion, nor did it result in the dysfunction of lysosomes. Instead, S130 might attenuate the delipidation of LC3-II on the autolysosomes to suppress the recycling of LC3-I, which normally occurs after LC3-II cleavage by ATG4B. Intriguingly, S130 induced cell death, which was accompanied with autophagy stress and could be further exacerbated by nutrient deprivation. Such cytotoxicity could be partially reversed by enhancing ATG4B activity. Finally, we found that S130 was distributed in tumor tissues in vivo and was also effective in arresting the growth of colorectal cancer cells. Thus, this study indicates that ATG4B is a potential anticancer target and S130 might be a novel small-molecule candidate for future cancer therapy.

During times of stress, autophagy is a cellular process that enables cells to reclaim damaged components by a controlled recycling pathway. This mechanism for cellular catabolism is dysregulated in cancer, with evidence indicating that cancer cells rely on autophagy in the hypoxic and nutrient-poor microenvironment of solid tumors. Mounting evidence suggests that autophagy has a role in the resistance of tumors to standard-of-care (SOC) therapies. Therefore, there is significant interest in the discovery of small molecules that can safely modulate autophagy. In this review, we describe recent advances in the identification of new pharmacological compounds that modulate autophagy, with a focus on their mode of action, value as probe compounds, and validation as potential therapeutics., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ischemic heart disease (IHD) is a significant cause of cardiovascular diseases. MicroRNAs (miRNAs) have been thought to be critical regulators in the heart diseases. The present study was aimed to investigate the effect of miR-129-5p on the autophagy and apoptosis by targeting ATG14 as well as how miR-129-5p worked through the PI3K/AKT/mTOR signaling pathway in H 2 O 2 -induced H9c2 cells. H9c2 cells were induced by H 2 O 2 , after which the expression of miR-129-5p was decreased. Reverse transcription-quantitative polymerase chain reaction (qRT-PCR) was performed to detect the expression level of miR-129-5p in H9c2 cells. In addition, the expression of miR-129-5p and ATG14 were overexpressed or down-regulated after transfection. The transfection efficiency was verified by qRT-PCR. Cell viability, cell apoptosis, and the expression of autophagy and apoptosis-related proteins were determined by CCK-8, flow cytometry and western blotting, respectively. Furthermore, GFP fusion protein analysis was used to detect the expression level of LC3II which was related to autophagy. As a result, cell viability was decreased and cell autophagy was increased in H 2 O 2 -induced H9c2 cells. MiR-129-5p overexpression inhibited cell injury caused by H 2 O 2 in H9c2 cells which was certified by the increased cell viability and decreased cell autophagy and apoptosis. In addition, ATG14 was demonstrated to be a target of miR-129-5p which inhibited cell injury by down-regulation of ATG14. Moreover, phosphorylation of PI3K/AKT/mTOR pathway was activated by miR-129-5p overexpression or ATG14 inhibition to alleviate the autophagy and apoptosis in H 2 O 2 -induced H9c2 cells. In conclusion, this study indicated that miR-129-5p inhibited autophagy and apoptosis in H 2 O 2 -induced H9c2 cells partly by down-regulation of ATG14 through the activation of PI3K/AKT/mTOR pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4-28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts.

Atg4 is essential for autophagosome formation and Atg8 recycle with the function of processing the precursor and the lipidated Atg8-family proteins. Abnormal autophagic activity is involved in a variety of pathophysiological diseases and ATG4B is of interest as a potential therapeutic target due to its key roles in autophagy process. So ATG4B inhibitors are highly needed. FMK-9a is the most potent inhibitor reported so far. In this study, we confirmed FMK-9a could suppress ATG4B activity in vitro and in cells, with an IC50 of 260 nM. Besides, FMK-9a could also attenuate the process of cleavage of pro-LC3 and the delipidation of LC3-PE. Importantly, FMK-9a could induce autophagy both in HeLa and MEF cells regardless of its inhibition on ATG4B activity. Moreover, FMK-9a induced autophagy required FIP200 and ATG5. In conclusion, we demonstrated that ATG4B inhibitor FMK-9a induces autophagy independent on its enzyme inhibition. Thus, FMK-9a may plays multiple roles in autophagy process and cannot simply take it as an ATG4B inhibitor., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The activation of androgen receptor (AR) signaling plays an essential role in both prostate stromal cells and epithelial cells during the development of benign prostatic hyperplasia (BPH). Here we demonstrated that androgen ablation after 5α-reductase inhibitor (5-ARI) treatment induced autophagy in prostate stromal fibroblasts inhibiting cell apoptosis. In addition, we found that ATG9A expression was increased after androgen ablation, which facilitated autophagic flux development. Knockdown of ATG9A not only inhibited autophagy notably in prostate stromal fibroblasts, but also reduced the volumes of prostate stromal fibroblast and epithelial cell recombinant grafts in nude mice. In conclusion, our findings suggested that ATG9A upregulation after long-term 5-ARI treatment constitutes a possible mechanism of BPH progression. Thus, combined treatment with 5-ARI and autophagy inhibitory agents would reduce the risk of BPH progression.

The proteins involved in the autophagy (Atg) pathway have recently been considered promising targets for the development of new antimalarial drugs. In particular, inhibitors of the protein-protein interaction (PPI) between Atg3 and Atg8 of Plasmodium falciparum retarded the blood- and liver-stages of parasite growth. In this paper, we used computational techniques to design a new class of peptidomimetics mimicking the Atg3 interaction motif, which were then synthesized by click-chemistry. Surface plasmon resonance has been employed to measure the ability of these compounds to inhibit the Atg3-Atg8 reciprocal protein-protein interaction. Moreover, P. falciparum growth inhibition in red blood cell cultures was evaluated as well as the cyto-toxicity of the compounds.

Protozoan parasites, including the apicomplexan pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis), infect millions of people worldwide and represent major human disease burdens. Despite their prevalence, therapeutic strategies to treat infections caused by these parasites remain limited and are threatened by the emergence of drug resistance, highlighting the need for the identification of novel drug targets. Recently, homologues of the core autophagy proteins, including Atg8 and Atg3, were identified in many protozoan parasites. Importantly, components of the Atg8 conjugation system that facilitate the lipidation of Atg8 are required for both canonical and parasite-specific functions and are essential for parasite viability. Structural characterization of the P. falciparum Atg3-Atg8 (PfAtg3-Atg8) interaction has led to the identification of compounds that block this interaction. Additionally, many of these compounds inhibit P. falciparum growth in vitro , demonstrating the viability of this pathway as a drug target. Given the essential role of the Atg8 lipidation pathway in Toxoplasma , we sought to determine whether three PfAtg3-Atg8 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box exerted a similar inhibitory effect in Toxoplasma While all three inhibitors blocked Toxoplasma replication in vitro at submicromolar concentrations, they did not inhibit T. gondii Atg8 (TgAtg8) lipidation. Rather, high concentrations of two of these compounds induced TgAtg8 lipidation and fragmentation of the parasite mitochondrion, similar to the effects seen following starvation and monensin-induced autophagy. Additionally, we report that one of the PfAtg3-Atg8 interaction inhibitors induces Toxoplasma egress and provide evidence that this is mediated by an increase in intracellular calcium in response to drug treatment., (Copyright © 2018 American Society for Microbiology.)

Autophagy is a primordial eukaryotic pathway, which provides the immune system with multiple mechanisms for the elimination of invading pathogens including Mycobacterium tuberculosis (Mtb). As a consequence, Mtb has evolved different strategies to hijack the autophagy process. Given the crucial role of human primary dendritic cells (DC) in host immunity control, we characterized Mtb-DC interplay by studying the contribution of cellular microRNAs (miRNAs) in the post-transcriptional regulation of autophagy related genes. From the expression profile of de-regulated miRNAs obtained in Mtb-infected human DC, we identified 7 miRNAs whose expression was previously found to be altered in specimens of TB patients. Among them, gene ontology analysis showed that miR-155, miR-155* and miR-146a target mRNAs with a significant enrichment in biological processes linked to autophagy. Interestingly, miR-155 was significantly stimulated by live and virulent Mtb and enriched in polysome-associated RNA fraction, where actively translated mRNAs reside. The putative pair interaction among the E2 conjugating enzyme involved in LC3-lipidation and autophagosome formation-ATG3-and miR-155 arose by target prediction analysis, was confirmed by both luciferase reporter assay and Atg3 immunoblotting analysis of miR-155-transfected DC, which showed also a consistent Atg3 protein and LC3 lipidated form reduction. Late in infection, when miR-155 expression peaked, both the level of Atg3 and the number of LC3 puncta per cell (autophagosomes) decreased dramatically. In accordance, miR-155 silencing rescued autophagosome number in Mtb infected DC and enhanced autolysosome fusion, thereby supporting a previously unidentified role of the miR-155 as inhibitor of ATG3 expression. Taken together, our findings suggest how Mtb can manipulate cellular miRNA expression to regulate Atg3 for its own survival, and highlight the importance to develop novel therapeutic strategies against tuberculosis that would boost autophagy.

Background/aims: The purpose of our experiments was to investigate the targeting relationship of linc00515, miR-140-5p and ATG14 and to explore the roles of linc00515, miR-140-5p and ATG14 in autophagy and chemoresistance of melphalan-resistant multiple myeloma cells., Methods: Plasmids that could interfere with the expression of linc00515 and ATG14 were loaded into myeloma cells, which were cultured with melphalan. MTT assay and flow cytometry analysis were utilized to investigate the effect of linc00515, miR-140-5p and ATG14 on the resistance of myeloma cells. QRT-PCR was used to determine the levels of mRNAs. Western blot was utilized to explore the level of ATG14 and autophagy-related proteins. Dual luciferase assay was utilized to explore the targeting relationship between linc00515, miR-140-5p and ATG14. GFP LC3 fluorescence assay was conducted to study the autophagy of cells., Results: The expression of linc00515 and ATG14 were significantly higher in melphalan-resistant myeloma cells. Knockdown of linc00515 and ATG14 led to decreased autophagy and chemoresistance of melphalan-resistant myeloma cells. The forced expression of miR-140-5p suppressed autophagy and chemoresistance of melphalan-resistant myeloma cells., Conclusion: Linc00515 enhanced autophagy and chemoresistance of melphalan-resistant myeloma by directly inhibiting miR-140-5p, which elevated ATG14 level., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background: Tumor cells require proficient autophagy to meet high metabolic demands and resist chemotherapy, which suggests that reducing autophagic flux might be an attractive route for cancer therapy. However, this theory in clinical cancer research remains controversial due to the limited number of drugs that specifically inhibit autophagy-related (ATG) proteins. Methods: We screened FDA-approved drugs using a novel platform that integrates computational docking and simulations as well as biochemical and cellular reporter assays to identify potential drugs that inhibit autophagy-required cysteine proteases of the ATG4 family. The effects of ATG4 inhibitors on autophagy and tumor suppression were examined using cell culture and a tumor xenograft mouse model. Results: Tioconazole was found to inhibit activities of ATG4A and ATG4B with an IC 50 of 1.3 µM and 1.8 µM, respectively. Further studies based on docking and molecular dynamics (MD) simulations supported that tioconazole can stably occupy the active site of ATG4 in its open form and transiently interact with the allosteric regulation site in LC3, which explained the experimentally observed obstruction of substrate binding and reduced autophagic flux in cells in the presence of tioconazole. Moreover, tioconazole diminished tumor cell viability and sensitized cancer cells to autophagy-inducing conditions, including starvation and treatment with chemotherapeutic agents. Conclusion: Tioconazole inhibited ATG4 and autophagy to enhance chemotherapeutic drug-induced cytotoxicity in cancer cell culture and tumor xenografts. These results suggest that the antifungal drug tioconazole might be repositioned as an anticancer drug or chemosensitizer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The investigation of autophagy particularly when observed during programmed cell death (PCD) is strongly based on the morphological features recorded with transmission electron microscope (TEM). Here we describe methods to induce and to inhibit autophagy in plants. Also some tips for obtaining better preservation of biological membranes, crucial for the investigation of autophagy, are provided together with information about plant autophagic mutants, use of antibodies and methods for 3D reconstruction of large membrane-bound objects that are commonly formed during autophagic processes leading to PCD in plants.

Autophagy is a cell survival mechanism hijacked by advanced tumors to endure a rough microenvironment. Late autophagy inhibitors such as (hydroxy)chloroquine have been used clinically to halt tumor progression with modest success. However, given the toxic nature of these compounds and their lack of specificity, novel targets should be considered. We recently identified a benzotropolone derivative that significantly inhibited the essential autophagy protein ATG4B. Therefore, we synthesized and tested additional benzotropolone compounds to identify a promising ATG4B inhibitor that impairs autophagy both in vitro and in vivo. A compound library containing 27 molecules with a benzotropolone backbone was synthesized and screened for inhibition of recombinant ATG4B. Depending on the benzotropolone compound, inhibition of recombinant ATG4B ranged from 3 to 82%. Active compounds were evaluated in cellular assays to confirm inhibition of ATG4B and suppression of autophagy. Seven compounds inhibited processing of the autophagy protein LC3 and autophagosome formation. Compound UAMC-2526 was selected for further in vivo use because of its fair plasma stability. This compound abolished autophagy both in nutrient-deprived GFP-LC3 mice and in CD1 -/- Foxn1nu mice bearing HT29 colorectal tumor xenografts. Moreover, addition of UAMC-2526 to the chemotherapy drug oxaliplatin significantly improved inhibition of tumor growth. Our data indicate that suppression of autophagy via ATG4B inhibition is a feasible strategy to augment existing chemotherapy efficacy and to halt tumor progression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Many human muscle wasting diseases are associated with abnormal nuclear localization. During metamorphosis in Drosophila melanogaster, multi-nucleated larval dorsal abdominal muscles either undergo cell death or are remodeled to temporary adult muscles. Muscle remodeling is associated with anti-polar nuclear migration and atrophy during early pupation followed by polar migration and muscle growth during late pupation. Muscle remodeling is a useful model to study genes involved in myonuclear migration. Previously, we showed that loss of Cathepsin-L inhibited anti-polar movements, while knockdown of autophagy-related genes affected nuclear positioning along the medial axis in late metamorphosis., Results: To compare the phenotypic effects of gene perturbations on nuclear migration more objectively, we developed new descriptors of myonuclear distribution. To obtain nuclear pattern features, we designed an algorithm to detect and track nuclear regions inside live muscles. Nuclear tracks were used to distinguish between fast moving nuclei associated with fragments of dead muscles (sarcolytes) and slow-moving nuclei inside remodelled muscles. Nuclear spatial pattern features, such as longitudinal (lonNS) and lateral nuclear spread (latNS), allowed us to compare nuclear migration during muscle remodelling in different genetic backgrounds. Anti-polar migration leads to a lonNS decrease. As expected, lack of myonuclear migration caused by the loss of Cp1 was correlated with a significantly lower lonNS decrease. Unexpectedly, the decrease in lonNS was significantly enhanced by Atg9, Atg5 and Atg18 silencing, indicating that the loss of autophagy promotes the migration and clustering of nuclei. Loss of autophagy also caused a scattering of nuclei along the lateral axis, leading to a two-row as opposed to single row distribution in control muscles. Increased latNS resulting from knockdown of Atg9 and Atg18 was correlated with increased muscle diameter, suggesting that the wider muscle fibre promotes lateral displacement of nuclei from the medial axis during polar migration., Conclusions: We developed new nuclear features to characterize the dynamics of nuclear distribution in time-lapse images of Drosophila metamorphosis. Image quantification improved our understanding of phenotypic abnormalities in nuclear distribution resulting from gene perturbations. Therefore, in vivo imaging and quantitative image analysis of Drosophila metamorphosis promise to provide novel insights into the relationship between muscle wasting and myonuclear positioning.

Autophagy is a catabolic mechanism to degrade cellular components to maintain cellular energy levels during starvation, a condition where PPARα may be activated. Here we report a reduced autophagic capacity in the liver following chronic activation of PPARα with fenofibrate (FB) in mice. Chronic administration of the PPARα agonist FB substantially reduced the levels of multiple autophagy proteins in the liver (Atg3, Agt4B, Atg5, Atg7 and beclin 1) which were associated with a decrease in the light chain LC3II/LC3I ratio and the accumulation of p62. This was concomitant with an increase in the expression of lipogenic proteins mSREBP1c, ACC, FAS and SCD1. These effects of FB were completely abolished in PPARα-/- mice but remained intact in mice with global deletion of FGF21, a key downstream mediator for PPARα-induced effects. Further studies showed that decreased the content of autophagy proteins by FB was associated with a significant reduction in the level of FoxO1, a transcriptional regulator of autophagic proteins, which occurred independently of both mTOR and Akt. These findings suggest that chronic stimulation of PPARα may suppress the autophagy capacity in the liver as a result of reduced content of a number of autophagy-associated proteins independent of FGF21.

Autophagy is an evolutionarily conserved homeostasis process through which aggregated proteins or damaged organelles are enveloped in a double-membrane structure called an autophagosome and then digested in a lysosome-dependent manner. Growing evidence suggests that malfunction of autophagy contributes to the pathogenesis of a variety of diseases, including cancer, viral infection, and neurodegeneration. However, autophagy is a complicated process, and understanding of the relevance of autophagy to disease is limited by lack of specific and potent autophagy modulators. ATG4B, a Cys-protease that cleaves ATG8 family proteins, such as LC3B, is a key protein in autophagosome formation and maturation process. A novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay measuring protease activity of ATG4B was developed, validated, and adapted into a high-throughput screening (HTS) format. HTS was then conducted with a Roche focus library of 57,000 compounds. After hit confirmation and a counterscreen to filter out fluorescence interference compounds, 267 hits were confirmed, constituting a hit rate of 0.49%. Furthermore, among 65 hits with an IC 50 < 50 µM, one compound mimics the LC3 peptide substrate (-TFG-). Chemistry modification based on this particular hit gave preliminary structure activity relationship (SAR) resulting in a compound with a 10-fold increase in potency. This compound forms a stable covalent bond with Cys74 of ATG4B in a 1:1 ratio as demonstrated by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Furthermore, this compound displayed cellular ATG4B inhibition activity. Overall, the novel TR-FRET ATG4B protease assay plus counterscreen assay provides a robust platform to identify ATG4B inhibitors, which would help to elucidate the mechanism of the autophagy pathway and offer opportunities for drug discovery.

Background: Drug resistance and disease progression are still the major obstacles in the treatment of chronic myeloid leukemia (CML). Increasing researches have demonstrated that autophagy becomes activated when cancer cells are subjected to chemotherapy, which is involved in the development of drug resistance. Therefore, combining chemotherapy with inhibition of autophagy serves as a new strategy in cancer treatment. Tigecycline is an antibiotic that has received attention as an anti-cancer agent due to its inhibitory effect on mitochondrial translation. However, whether combination of tigecycline with inhibition of autophagy could overcome drug resistance in CML remains unclear., Methods: We analyzed the biological and metabolic effect of tigecycline on CML primary cells and cell lines to investigate whether tigecycline could regulate autophagy in CML cells and whether coupling autophagy inhibition with treatment using tigecycline could affect the viabilities of drug-sensitive and drug-resistant CML cells., Results: Tigecycline inhibited the viabilities of CML primary cells and cell lines, including those that were drug-resistant. This occurred via the inhibition of mitochondrial biogenesis and the perturbation of cell metabolism, which resulted in apoptosis. Moreover, tigecycline induced autophagy by downregulating the PI3K-AKT-mTOR pathway. Additionally, combining tigecycline use with autophagy inhibition further promoted the anti-leukemic activity of tigecycline. We also observed that the anti-leukemic effect of tigecycline is selective. This is because the drug targeted leukemic cells but not normal cells, which is because of the differences in the mitochondrial biogenesis and metabolic characterization between the two cell types., Conclusions: Combining tigecycline use with autophagy inhibition is a promising approach for overcoming drug resistance in CML treatment.

MicroRNAs (miRNAs) have been the subject of recent attention as key regulatory factors in cell differentiation. In the current study, to explore the early signaling cascade of osteogenic differentiation of human induced pluripotent stem (hiPS) cells, we investigated miR-211 regulation and autophagy-related gene (Atg) signaling in osteogenic differentiation. In addition to reciprocal strong induction of miR-211 expression in differentiated cells following osteogenic differentiation, we found abundant Argonaute 3 bound to miR-211. There were also dramatic increases in the mRNA and protein levels of Atg14 together with increases in the amount of autophagosomes as well as autophagic fluxes. While transfection of a miR-211 inhibitor abrogated the induction of Atg14, autophagy events, osteoblast differentiation markers, and induction of calcification were suppressed markedly. Treatment with small interfering RNAs against Atg14 also suppressed the osteogenic differentiation medium (ODM)-induced increase in osteogenic differentiation. The osteogenic phenotype was inhibited by chloroquine (an autophagy inhibitor), but increased after treatment with rapamycin (an autophagy inducer). Taken together with our previous findings, we have revealed a unique sequential cascade involving miR-211 and Atg14 in ODM-induced differentiation of hiPS cells into osteoblast-like cells at a relatively early stage., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Autophagy is an evolutionarily conserved cellular process that primarily participates in lysosome-mediated protein degradation. Although autophagy is a cytoplasmic event, how epigenetic pathways are involved in the regulation of autophagy remains incompletely understood. Here, we found that H2B monoubiquitination (H2Bub1) is down-regulated in cells under starvation conditions and that the decrease in H2Bub1 results in the activation of autophagy. We also identified that the deubiquitinase USP44 is responsible for the starvation-induced decrease in H2Bub1. Furthermore, the changes in H2Bub1 affect the transcription of genes involved in the regulation of autophagy. Therefore, this study reveals a novel epigenetic pathway for the regulation of autophagy through H2Bub1.

Background/aims: Our previous microarray results identified numerous microRNAs (miRNAs), including miR-29b, that were differentially expressed in the serum of intracranial aneurysm (IA) patients. The current study aimed to investigate whether miR-29b downregulation in IA could promote the phenotypic modulation of vascular smooth muscle cells (VSMCs) involved in the pathogenesis of aneurysm by activating ATG14-mediated autophagy., Methods: First, the levels of miR-29b and autophagy related genes (ATGs) between IA patients and normal subjects were compared. Next, we modified the level of miR-29b via lentivirus particles in the VSMCs and examined the effects of miR-29b on proliferation, migration, and phenotypic modulation of VSMCs from a contractile phenotype to a synthetic phenotype, as well as the levels of autophagy. Finally, the binding of miR-29b to the 3'UTR of ATG14 mRNA and its effects on ATG14 expression were analysed by a luciferase reporter assay and Western blot, respectively., Results: The level of miR-29b was decreased, and autophagy markers were increased in the IA patients compared to that of the normal subjects. Knockdown of miR-29b significantly promoted VSMCs proliferation and migration and, more importantly, induced the phenotypic modulation associated with autophagy activation, whereas miR-29b overexpression showed the opposite effects. The luciferase reporter assay demonstrated that ATG14 was a functional target gene of miR-29b. Notably, knockdown of ATG14 by siRNA apparently abrogated miR-29b inhibition-mediated phenotypic modulation., Conclusion: Downregulation of miR-29b induced VSMCs phenotypic modulation by directly activating ATG14-mediated autophagy, which is associated with the formation, growth and rupture of IAs., (© 2017 The Author(s) Published by S. Karger AG, Basel.)

Background/aims: ATG4B is a cysteine protease required for autophagy, which is a cellular catabolic pathway involved in energy balance. ATG4B expression is elevated during tumor growth in certain types of cancer, suggesting that ATG4B is an attractive target for cancer therapy. However, little is known about the mechanisms through which ATG4B deprivation suppresses the growth of cancer cells., Methods: Cancer cells were transfected with either siRNA against ATG4B or an expression vector encoding wild-type ATG4BWT or encoding catalytic mutant ATG4BC74A to determine cell cycle progression by propidium iodide staining or by BrdU incorporation assay using flow cytometry. The GFP-MAP1LC3-II puncta and protein levels in the cells were determined by immunofluorescence and immunoblotting, respectively., Results: Knockdown of ATG4B blocked cell proliferation, particularly at the G1-S phase transition, in various cancer cells. Moreover, knockdown of ATG4B or overexpression of the ATG4BC74A catalytic mutant reduced both autophagic flux and ATP levels and increased AMP-activated protein kinase (AMPK) phosphorylation in the cancer cells. Nevertheless, knockdown of ATG4B had only a minor effect on AMPK activation and G1 phase arrest in liver kinase B1 (LKB1)-deficient or AMPK-inhibited cancer cells., Conclusion: These results imply that targeting ATG4B might inhibit autophagy and trigger the LKB1-AMPK energy-sensing pathway, resulting in tumor growth suppression., (© 2017 The Author(s). Published by S. Karger AG, Basel.)