Autophagy activation, lipotoxicity and lysosomal membrane permeabilization synergize to promote pimozide- and loperamide-induced glioma cell death.

Increasing evidence suggests that induction of lethal macroautophagy/autophagy carries potential significance for the treatment of glioblastoma (GBM). In continuation of previous work, we demonstrate that pimozide and loperamide trigger an ATG5- and ATG7 (autophagy related 5 and 7)-dependent type of cell death that is significantly reduced with cathepsin inhibitors and the lipid reactive oxygen species (ROS) scavenger α-tocopherol in MZ-54 GBM cells. Global proteomic analysis after treatment with both drugs also revealed an increase of proteins related to lipid and cholesterol metabolic processes. These changes were accompanied by a massive accumulation of cholesterol and other lipids in the lysosomal compartment, indicative of impaired lipid transport/degradation. In line with these observations, pimozide and loperamide treatment were associated with a pronounced increase of bioactive sphingolipids including ceramides, glucosylceramides and sphingoid bases measured by targeted lipidomic analysis. Furthermore, pimozide and loperamide inhibited the activity of SMPD1/ASM (sphingomyelin phosphodiesterase 1) and promoted induction of lysosomal membrane permeabilization (LMP), as well as release of CTSB (cathepsin B) into the cytosol in MZ-54 wild-type (WT) cells. Whereas LMP and cell death were significantly attenuated in ATG5 and ATG7 knockout (KO) cells, both events were enhanced by depletion of the lysophagy receptor VCP (valosin containing protein), supporting a pro-survival function of lysophagy under these conditions. Collectively, our data suggest that pimozide and loperamide-driven autophagy and lipotoxicity synergize to induce LMP and cell death. The results also support the notion that simultaneous overactivation of autophagy and induction of LMP represents a promising approach for the treatment of GBM.Abbreviations: ACD: autophagic cell death; AKT1: AKT serine/threonine kinase 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; CERS1: ceramide synthase 1; CTSB: cathepsin B; CYBB/NOX2: cytochrome b-245 beta chain; ER: endoplasmatic reticulum; FBS: fetal bovine serum; GBM: glioblastoma; GO: gene ontology; HTR7/5-HT7: 5-hydroxytryptamine receptor 7; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAP: LC3-associated phagocytosis; LMP: lysosomal membrane permeabilization; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; RB1CC1: RB1 inducible coiled-coil 1; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SMPD1/ASM: sphingomyelin phosphodiesterase 1; VCP/p97: valosin containing protein; WT: wild-type.

Discovery and optimization of pyrazolopyrimidine sulfamates as ATG7 inhibitors.

Autophagy is postulated to be required by cancer cells to survive periods of metabolic and/or hypoxic stress. ATG7 is the E1 enzyme that is required for activation of Ubl conjugation pathways involved in autophagosome formation. This article describes the design and optimization of pyrazolopyrimidine sulfamate compounds as potent and selective inhibitors of ATG7. Cellular levels of the autophagy markers, LC3B and NBR1, are regulated following treatment with these compounds.

LncRNA SNHG3 promotes autophagy-induced neuronal cell apoptosis by acting as a ceRNA for miR-485 to up-regulate ATG7 expression.

Long non-coding RNAs (lncRNAs) are bound up with various human diseases. However, their roles in brain ischemia-reperfusion (I/R) injury remain largely unknown. This study aimed to reveal the potential mechanism of LncRNA SNHG3 on autophagy-induced neuronal cell apoptosis in the brain I/R injury. LncRNA SNHG3 and miR-485 or autophagy markers LC3II/I and Beclin-1 expressions were detected by qRT-PCR or Western blot and the apoptosis of N2a cells was analyzed by flow cytometry. Besides, the interactions between LncRNA SNHG3 and miR-485, miR-485 and ATG7 were validated by RNA pull-down and dual-luciferase reporter system assays. After the Oxygen and Glucose Deprivation (OGD) treatment of N2a cells transfected with pcDNA-SNHG3, pcDNA-SNHG3 + miR-485 mimic for 6 h, 1 mM autophagy inhibitor 3-MA was added and reoxygenated for 24 h, the effect of LncRNA SNHG3 on the autophagy-induced neuronal cell apoptosis was measured by Western blot and flow cytometry. LncRNA SNHG3 was highly expressed in the mouse model of transient middle cerebral artery occlusion and cell model of Oxygen and Glucose Deprivation/Reperfusion, while miR-485 was lowly expressed. Furthermore, miR-485 negatively regulated the luciferase activities of LncRNA SNHG3 and ATG7. After the OGD treatment of N2a cells transfected with pcDNA-SNHG3, pcDNA-SNHG3 + miR-485 mimic for 6 h, 1 mM 3-MA was added and reoxygenated for 24 h, the overexpression of LncRNA SNHG3 raised the ratio of LC3-II/LC3-I and Beclin-1 expression and boosted the apoptosis of N2a cells, while these effects were reversed after the transfection of miR-485 mimic. In general, our data expounded that the interference with LncRNA SNHG3 improved brain I/R injury by up-regulating miR-485 and down-regulating ATG7 to restrain autophagy and neuronal cell apoptosis.

miR-375 Inhibits Autophagy and Further Promotes Inflammation and Apoptosis of Acinar Cells by Targeting ATG7.

OBJECTIVES: MicroRNAs have been considered to be closely related with the development of severe acute pancreatitis (SAP), and microRNA-375 (miR-375) was believed to be a marker of SAP. We aim to investigate the role of miR-375 in regulating SP. METHODS: Cerulein and lipopolysaccharide were used to establish the models of SAP. AR42J cell line was chosen for study in vitro. Flow cytometry was applied for assessing apoptosis. The contents of inflammatory factors were detected with related enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction assays. Hematoxylin and eosin staining was applied to observe the pathological changes of pancreatic tissues. Immunohistochemistry analysis was conducted for investigating the expression of light chain 3. RESULTS: The level of miR-375 in pancreatitis tissues and cell lines was upregulated. Overexpression of miR-375 promoted inflammation and the apoptosis of acinar cells through inhibiting autophagy. The binding site between miR-375 and ATG7 was identified, and miR-375 could directly regulate the ATG7. microRNA-375 suppressed autophagy and promoted inflammation and the apoptosis of acinar cells via targeting ATG7. CONCLUSIONS: We proved that miR-375 could inhibit autophagy and promote inflammation and the apoptosis of acinar cells through regulating ATG7. This study first proves that miR-375 modulates the development of SAP through targeting ATG7.

FOXM1 contributes to docetaxel resistance in castration-resistant prostate cancer by inducing AMPK/mTOR-mediated autophagy.

Docetaxel-mediated chemotherapy is the first line therapy for metastatic castration-resistant prostate cancer (CRPC) patients, but its therapeutic benefit is limited by the development of resistance. Although Forkhead box protein M1 (FOXM1) has been implicated in prostate tumorigenesis and metastasis, its role in docetaxel resistance has not been studied. Here, we showed that FOXM1 expression was upregulated in the docetaxel resistant CRPC cell lines (PC3-DR and VCaP-DR) and knockdown of FOXM1 sensitized the cells to docetaxel both in vitro and in vivo. In addition, autophagy was found to be significantly enhanced in resistant cells. Moreover, FOXM1 overexpression cells showed increased autophagic flux and higher numbers of autophagosomes. Knockdown of ATG7, beclin-1 or cotreatment with chloroquine, partly restored sensitivity to docetaxel in the FOXM1-overexpressing cells. Mechanistically, FOXM1 targeted AMPK/mTOR to activate the autophagy pathway and altered docetaxel response in CRPC. These findings identify the role of FOXM1 as well as the mechanism underlying FOXM1 action in docetaxel sensitivity and may, therefore, aid in design of CRPC therapies.

Atg7 Silencing Inhibits Laminin-5 Expression to Suppress Tube Formation by Brain Endothelial Cells.

Cerebral angiogenesis is a key event during brain development and recovery from brain injury. We previously demonstrated that Atg7 knockout impaired angiogenesis in the mouse brain. However, the role of Atg7 in angiogenesis is not completely understood. In this study, we used human brain microvascular endothelial cells (HBMECs) to investigate the mechanism of Atg7-regulated cerebral angiogenesis. We found that Atg7 depletion specifically diminished the expression of the ß3 and γ2 chains of laminin-5, a major component of the extracellular matrix. In contrast, autophagy inhibitors did not affect laminin-5 expression, suggesting that Atg7-regulated laminin-5 expression is autophagy-independent. We also found that Atg7-regulated laminin-5 expression occurred at the transcriptional level through NF-κB signaling. Exogenous laminin-5 or the NF-κB agonist betulinic acid effectively rescued tube formation by Atg7-deficient HBMECs. Taken together, our study identified a novel mechanism by which Atg7 regulates laminin-5 expression via NF-κB to modulate tube formation by brain endothelial cells during cerebral angiogenesis. Anat Rec, 302:2255-2260, 2019. © 2019 American Association for Anatomy.

Autophagy facilitates the release of immunogenic signals following chemotherapy in 3D models of mesothelioma.

We have previously shown that nearly half of mesothelioma patients have tumors with low autophagy and that these patients have a significantly worse outcome than those with high autophagy. We hypothesized that autophagy may be beneficial by facilitating immunogenic cell death (ICD) of tumor cells following chemotherapy. An important hallmark of ICD is that death of tumor cells is preceded or accompanied by the release of damage-associated molecular pattern molecules (DAMPs), which then can stimulate an antitumor immune response. Therefore, we measured how autophagy affected the release of three major DAMPs: high mobility group box 1 (HMGB1), ATP, and calreticulin following chemotherapy. We found that autophagy in three-dimensional (3D) models with low autophagy at baseline could be upregulated with the cell-permeant Tat-BECN1 peptide and confirmed that autophagy in 3D models with high autophagy at baseline could be inhibited with MRT 68921 or ATG7 RNAi, as we have previously shown. In in vitro 3D spheroids, we found that, when autophagy was high or upregulated, DAMPs were released following chemotherapy; however, when autophagy was low or inhibited, DAMPs release was significantly impaired. Similarly, in ex vivo tumors, when autophagy was high or upregulated, HMGB1 was released following chemotherapy but, when autophagy was low, HMGB1 release was not seen. We conclude that autophagy can be upregulated in at least some tumors with low autophagy and that upregulation of autophagy can restore the release of DAMPs following chemotherapy. Autophagy may be necessary for ICD in this tumor.

miR-27a-5p Attenuates Hypoxia-induced Rat Cardiomyocyte Injury by Inhibiting Atg7.

Acute myocardial infarction (AMI) is an ischemic heart disease with high mortality worldwide. AMI triggers a hypoxic microenvironment and induces extensive myocardial injury, including autophagy and apoptosis. MiRNAs, which are a class of posttranscriptional regulators, have been shown to be involved in the development of ischemic heart diseases. We have previously reported that hypoxia significantly alters the miRNA transcriptome in rat cardiomyoblast cells (H9c2), including miR-27a-5p. In the present study, we further investigated the potential function of miR-27a-5p in the cardiomyocyte response to hypoxia, and showed that miR-27a-5p expression was downregulated in the H9c2 cells at different hypoxia-exposed timepoints and the myocardium of a rat AMI model. Follow-up experiments revealed that miR-27a-5p attenuated hypoxia-induced cardiomyocyte injury by regulating autophagy and apoptosis via Atg7, which partly elucidated the anti-hypoxic injury effects of miR-27a-5p. Taken together, this study shows that miR-27a-5p has a cardioprotective effect on hypoxia-induced H9c2 cell injury, suggesting it may be a novel target for the treatment of hypoxia-related heart diseases.

Selective cell death of latently HIV-infected CD4+ T cells mediated by autosis inducing nanopeptides.

Despite significant advances in the treatment of human immunodeficiency virus type-1 (HIV) infection, antiretroviral therapy only suppresses viral replication but is unable to eliminate infection. Thus, discontinuation of antiretrovirals results in viral reactivation and disease progression. A major reservoir of HIV latent infection resides in resting central memory CD4+ T cells (TCM) that escape clearance by current therapeutic regimens and will require novel strategies for elimination. Here, we evaluated the therapeutic potential of autophagy-inducing peptides, Tat-Beclin 1 and Tat-vFLIP-α2, which can induce a novel Na+/K+-ATPase dependent form of cell death (autosis), to kill latently HIV-infected TCM while preventing virologic rebound. In this study, we encapsulated autophagy inducing peptides into biodegradable lipid-coated hybrid PLGA (poly lactic-co-glycolic acid) nanoparticles for controlled intracellular delivery. A single dose of nanopeptides was found to eliminate latent HIV infection in an in vitro primary model of HIV latency and ex vivo using resting CD4+ T cells obtained from peripheral blood mononuclear cells of HIV-infected patients on antiretroviral with fully suppressed virus for greater than 12 months. Notably, increased LC3B lipidation, SQSTM1/p62 degradation and Na+/K+-ATPase activity characteristic of autosis, were detected in nanopeptide treated latently HIV-infected cells compared to untreated uninfected or infected cells. Nanopeptide-induced cell death could be reversed by knockdown of autophagy proteins, ATG5 and ATG7, and inhibition or knockdown of Na+/K+-ATPase. Importantly, viral rebound was not detected following the induction of the Na+/K+-ATPase dependent form of cell death induced by the Tat-Beclin 1 and Tat-vFLIP-α2 nanopeptides. These findings provide a novel strategy to eradicate HIV latently infected resting memory CD4+ T cells, the major reservoir of HIV latency, through the induction of Na+/K+-ATPase dependent autophagy, while preventing reactivation of virus and new infection of uninfected bystander cells.

AKT inhibition-mediated dephosphorylation of TFE3 promotes overactive autophagy independent of MTORC1 in cadmium-exposed bone mesenchymal stem cells.

Cadmium (Cd) is a toxic metal that is widely found in numerous environmental matrices and induces serious adverse effects in various organs and tissues. Bone tissue seems to be a crucial target of Cd contamination. Macroautophagy/autophagy has been proposed to play a pivotal role in Cd-mediated bone toxicity. However, the mechanisms that underlie Cd-induced autophagy are not yet completely understood. We demonstrated that Cd treatment increased autophagic flux and inhibition of the autophagic process using Atg7 gene silencing blocked the Cd-induced mesenchymal stem cell death. Mechanistically, Cd activated nuclear translocation of TFE3 but not that of TFEB or MITF, which contributed to the expression of autophagy-related genes and lysosomal biogenesis. Specifically, Cd decreased expression of phospho-AKT (Ser473). The reduction in AKT activity led to dephosphorylation of cytosolic TFE3 at Ser565 and promoted TFE3 nuclear translocation independently of MTORC1. Notably, Cd treatment increased the activity of PPP3/calcineurin, and pharmacological inhibition of PPP3/calcineurin with FK506 suppressed AKT dephosphorylation and TFE3 activity. These results suggest that PPP3/calcineurin negatively regulates AKT phosphorylation and is involved in Cd-induced TFE3-dependent autophagy. Modulation of the PPP3/calcineurin-AKT-TFE3 autophagic-lysosomal machinery may offer novel therapeutic approaches for the treatment of Cd-induced bone damage. Abbreviations: ACTB: actin: beta; AKT: thymoma viral proto-oncogene; AMPK: AMP-activated protein kinase; ATG: autophagy related; Baf A1: bafilomycin A1; Cd: cadmium; FOXO3: forkhead box O3; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MITF: melanogenesis associated transcription factor; MSC: mesenchymal stem sell; MTORC1: mechanistic target of rapamycin kinase complex 1; RPS6KB1: ribosomal protein S6 kinase: polypeptide 1; SGK1: serum/glucocorticoid regulated kinase 1; SQSTM1/p62: sequestosome 1;TFE3: transcription factor E3; TFEB: transcription factor EB; TFEC: transcription factor EC.

Atg7 mediates renal tubular cell apoptosis in vancomycin nephrotoxicity through activation of PKC-δ.

Recent studies have shown that autophagy exhibits a renoprotective role in various models of acute kidney injury (AKI). However, its role in vancomycin (Van)-induced AKI remains largely unclarified. This study was the first to indicate that autophagy was rapidly activated in both human kidney-2 cells and renal tissues, and mammalian target of rapamycin (mTOR) was inactivated via the suppression of ERK1/2 and mTOR during Van treatment. Interestingly, for both in vitro and in vivo experiments, the suppression of autophagy via chloroquine and PT-Atg7-KO significantly ameliorated Van-induced kidney injury and renal tubular cell apoptosis. Global gene expression analysis indicated that the expression levels of 6159 genes were induced by Van treatment in the kidney cortical tissues of PT-Atg7 wild-type mice, and 18 of them were notably suppressed in PT-Atg7-KO mice. These 18 genes were further classified as programmed cell death, protein binding, signal transduction, E3 ubiquitin ligase, nucleoside diphosphate kinase activity, and E1-like activating enzyme. Unexpectedly, following Van treatment, PKC-δ expression was found to be highest among the 4 genes related to cell death, which was remarkably suppressed in vitro and in PT-Atg7-KO mice. In addition, Atg7 could induce renal cell apoptosis during Van treatment via binding to PKC-δ. Likewise, the inhibition of PKCδ ameliorated Van-induced apoptosis in human kidney-2 cells and kidney tissues. Furthermore, the data showed that PT-Atg7-KO exerted a renoprotective effect against Van-induced nephrotoxicity, but this effect was lost after injection with myc-tagged PKCδ. Taken altogether, these results indicate that Van induces autophagy by suppressing the activation of the ERK1/2 and mTOR signaling pathway. In addition, Atg7 mediates Van-induced AKI through the activation of PKCδ. In sum, autophagy inhibition may serve as a novel therapeutic target for treating nephrotoxic AKI induced by Van.-Xu, X., Pan, J., Li, H., Li, X., Fang, F., Wu, D., Zhou, Y., Zheng, P., Xiong, L., Zhang, D. Atg7 mediates renal tubular cell apoptosis in vancomycin nephrotoxicity through activation of PKC-δ.

AMPK promotes survival and adipogenesis of ischemia-challenged ADSCs in an autophagy-dependent manner.

Some studies have shown that transplanted fat tissues usually cannot survive for long if adipose-derived stem cells (ADSCs) are removed from the tissues in advance. It is more meaningful to explore the mechanism mediating survival and differentiation of ADSCs in the transplanted microenvironment. AMP-activated protein kinase (AMPK) has been shown to be one of the energy receptors that regulate many aspects of cellular metabolism. AMPK activation has been implicated in models of adult ischemic injury, but the mechanism and the regulating effects of AMPK on survival and adipogenesis of transplanted ADSCs are still little known. In this study, we simulated the transplanted microenvironment using oxygen-glucose deprivation (OGD) to test the survival and adipogenesis of ADSCs. We found that OGD treatment triggered significant apoptosis and promoted autophagy. Simultaneously, OGD hindered the differentiation of ADSCs into mature adipocytes. After inhibiting AMPK, the OGD-induced apoptosis rate increased but autophagy was inhibited. The adipogenesis level also decreased. To show that the effects of AMPK on apoptosis and adipogenesis were autophagy-dependent, we pre-inhibited or pre-promoted autophagy with siATG7 or rapamycin while blocking AMPK. We found that inhibiting or improving autophagy exacerbated or alleviated the role of AMPK prohibition in apoptosis and adipogenesis. Furthermore, we showed that AMPK inhibition significantly lowered ULK1 activity but promoted mTOR activity, so that to inhibit autophagy. Our study shows that AMPK plays a protective role in maintaining survival and adipogenesis of OGD-challenged ADSCs partly by positively regulating autophagy. AMPK positively regulates autophagy by inhibiting mTOR but promoting ULK1 activity in OGD condition.

AMPK-dependent autophagy upregulation serves as a survival mechanism in response to Tumor Treating Fields (TTFields).

Tumor Treating Fields (TTFields), an approved treatment modality for glioblastoma, are delivered via non-invasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application leads to abnormal mitosis, aneuploidy, and increased cell granularity, which are often associated with enhancement of autophagy. In this work, we evaluated whether TTFields effected the regulation of autophagy in glioma cells. We found that autophagy is upregulated in glioma cells treated with TTFields as demonstrated by immunoblot analysis of the lipidated microtubule-associated protein light chain 3 (LC3-II). Fluorescence and transmission electron microscopy demonstrated the presence of LC3 puncta and typical autophagosome-like structures in TTFields-treated cells. Utilizing time-lapse microscopy, we found that the significant increase in the formation of LC3 puncta was specific to cells that divided during TTFields application. Evaluation of selected cell stress parameters revealed an increase in the expression of the endoplasmic reticulum (ER) stress marker GRP78 and decreased intracellular ATP levels, both of which are indicative of increased proteotoxic stress. Pathway analysis demonstrated that TTFields-induced upregulation of autophagy is dependent on AMP-activated protein kinase (AMPK) activation. Depletion of AMPK or autophagy-related protein 7 (ATG7) inhibited the upregulation of autophagy in response to TTFields, as well as sensitized cells to the treatment, suggesting that cancer cells utilize autophagy as a resistance mechanism to TTFields. Combining TTFields with the autophagy inhibitor chloroquine (CQ) resulted in a significant dose-dependent reduction in cell growth compared with either TTFields or CQ alone. These results suggest that dividing cells upregulate autophagy in response to aneuploidy and ER stress induced by TTFields, and that AMPK serves as a key regulator of this process.

Inhibition of glioma growth by flavokawain B is mediated through endoplasmic reticulum stress induced autophagy.

Flavokawain B (FKB), a natural kava chalcone, displays potent antitumor activity in various types of cancer. The mechanism of action, however, remains unclear. Here, we evaluated the efficacy of FKB in the treatment of human glioblastoma multiforme (GBM) as well as the molecular basis for its inhibitory effects in cancer. Approximately 60% of GBM cells became senescent after treatment with FKB as assessed in the senescence-associated (SA)-GLB1/SA-ß-galactosidase assay. The cellular process of autophagy potentially contributed to the establishment of senescence. Transmission electron microscopy revealed the formation of autophagic vesicles under FKB treatment, and MAP1LC3B (microtubule associated protein 1 light chain 3 beta)-II was increased. Transfection of ATG5 or ATG7 small interfering RNAs (siRNAs) inhibited FKB-induced autophagy in U251 cells. Western blot revealed that molecular components of the endoplasmic reticulum stress pathway were activated, including ATF4 (activating transcription factor 4) and DDIT3 (DNA damage inducible transcript 3), while levels of TRIB3 (tribbles pseudokinase 3) increased. In addition, based on the phosphorylation status, the AKT-MTOR-RPS6KB1 pathway was inhibited, which induced autophagy in GBM cells. Inhibition of autophagy by autophagy inhibitors 3-methyladenine and chloroquine or knockdown of ATG5 or ATG7 caused FKB-treated U251 cells to switch from senescence to apoptosis. Finally, knockdown of ATG5 or treatment with chloroquine in combination with FKB, significantly inhibited tumor growth in vivo. Our results demonstrated that FKB induced protective autophagy through the ATF4-DDIT3-TRIB3-AKT-MTOR-RPS6KB1 signaling pathway in GBM cells, indicating that the combination treatment of FKB with autophagy inhibitors may potentially be an effective therapeutic strategy for GBM. ABBREVIATIONS: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATF4: activating transcription factor 4; ATG: autophagy related; CASP3: caspase 3; CCK-8: cell counting kit-8; CDKN1A: cyclin-dependent kinase inhibitor 1A; CQ: chloroquine; DDIT3: DNA damage inducible transcript 3; DMEM: Dulbecco's modified Eagle's medium; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; FKB: flavokawain B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBM: glioblastoma multiforme; GFP: green fluorescent protein; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase; 1RPS6KB1: ribosomal protein S6 kinase B1; SA-GLB1: senescence-associated galactosidase beta 1; siRNA: short interfering RNA; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TRIB3: tribbles pseudokinase 3; TUNEL: deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Interaction of E3 Ubiquitin Ligase MARCH7 with Long Noncoding RNA MALAT1 and Autophagy-Related Protein ATG7 Promotes Autophagy and Invasion in Ovarian Cancer.

BACKGROUND/AIMS: Ubiquitin E3 ligase MARCH7 plays an important role in T cell proliferation and neuronal development. But its role in ovarian cancer remains unclear. This study aimed to investigate the role of Ubiquitin E3 ligase MARCH7 in ovarian cancer. METHODS: Real-time PCR, immunohistochemistry and western blotting analysis were performed to determine the expression of MARCH7, MALAT1 and ATG7 in ovarian cancer cell lines and clinical specimens. The role of MARCH7 in maintaining ovarian cancer malignant phenotype was examined by Wound healing assay, Matrigel invasion assays and Mouse orthotopic xenograft model. Luciferase reporter assay, western blot analysis and ChIP assay were used to determine whether MARCH7 activates TGF-ß-smad2/3 pathway by interacting with TGFßR2. RESULTS: MARCH7 interacted with MALAT1 by miR-200a (microRNA-200a). MARCH7 may function as a competing endogenous RNA (ceRNA) to regulate the expression of ATG7 by competing with miR-200a. MARCH7 regulated TGF-ß-smad2/3 pathway by interacting with TGFßR2. Inhibition of TGF-ß-smad2/3 pathway downregulated MARCH7, MALAT1 and ATG7. MiR-200a regulated TGF-ß induced autophagy, invasion and metastasis of SKOV3 cells by targeting MARCH7. MARCH7 silencing inhibited autophagy invasion and metastasis of SKOV3 cells both in vitro and in vivo. In contrast, MARCH7 overexpression promoted TGF-ß induced autophagy, invasion and metastasis of A2780 cells in vitro by depending on MALAT1 and ATG7. We also found that TGF-ß-smad2/3 pathway regulated MARCH7 and ATG7 through MALAT1. CONCLUSIONS: These findings suggested that TGFßR2-Smad2/3-MALAT1/MARCH7/ATG7 feedback loop mediated autophagy, migration and invasion in ovarian cancer.

Autophagy promotes the survival of dormant breast cancer cells and metastatic tumour recurrence.

Cancer recurrence after initial diagnosis and treatment is a major cause of breast cancer (BC) mortality, which results from the metastatic outbreak of dormant tumour cells. Alterations in the tumour microenvironment can trigger signalling pathways in dormant cells leading to their proliferation. However, processes involved in the initial and the long-term survival of disseminated dormant BC cells remain largely unknown. Here we show that autophagy is a critical mechanism for the survival of disseminated dormant BC cells. Pharmacologic or genetic inhibition of autophagy in dormant BC cells results in significantly decreased cell survival and metastatic burden in mouse and human 3D in vitro and in vivo preclinical models of dormancy. In vivo experiments identify autophagy gene autophagy-related 7 (ATG7) to be essential for autophagy activation. Mechanistically, inhibition of the autophagic flux in dormant BC cells leads to the accumulation of damaged mitochondria and reactive oxygen species (ROS), resulting in cell apoptosis.

The Effect of and Mechanism Underlying Autophagy in Hepatocellular Carcinoma Induced by CH12, a Monoclonal Antibody Directed Against Epidermal Growth Factor Receptor Variant III.

BACKGROUND/AIMS: Epidermal growth factor receptor variant III (EGFRvIII), the most frequent EGFR variant, is constitutively activated without binding to EGF and is correlated with a poor prognosis. CH12, a human-mouse chimeric monoclonal antibody, has been developed in our laboratory and selectively binds to overexpressed EGFR and EGFRvIII. A previous study had reported that EGFR could influence autophagic activity, and autophagy is closely related to tumor development and the response to drug therapy. In this study, we aimed to elucidate the effect of CH12 on autophagy and efficacy of combining CH12 with an autophagy inhibitor against EGFRvIII-positive tumors. METHODS: EGFRvIII was overexpressed in liver cancer, glioblastoma and breast cancer, and the change in the autophagy-relevant protein levels was analyzed by western blot assays, LC3 punctate aggregation was analyzed by immunofluorescence. The interaction of Beclin-1 and Rubicon was assessed by co-immunoprecipitation (Co-IP) after CH12 treatment. The efficacy of ATG7 or Beclin-1 siRNA in combination with CH12 in Huh-7-EGFRvIII cells was assessed by CCK-8 assays. The autophagy and apoptosis signaling events in Huh-7-EGFRvIII cells upon treatment with control, CH12, siRNA or combination for 48 h were assessed by western blot assays. RESULTS: Our results showed that, in cancer cell lines overexpressing EGFRvIII, only the liver cancer cell lines Huh-7 and PLC/PRF/5 suggested autophagy activation. We then investigated the mechanism of autophagy activation after EGFRvIII overexpression. The results showed that EGFRvIII interacted with Rubicon, an autophagy inhibition protein, and released Beclin-1 to form the inducer complex, thus contributing to autophagy. In addition, CH12, via inhibiting the phosphorylation of EGFRvIII, promoted the interaction of EGFRvIII with Rubicon, further inducing autophagy. In vitro assays suggested that knocking down the expression of the key proteins ATG7 or Beclin-1 in the autophagy pathway with siRNA inhibits tumor cell proliferation. Combining autophagy-related proteins 7 (ATG7) or Beclin-1 siRNA with CH12 in Huh-7-EGFRvIII cells showed better inhibition of cell proliferation. CONCLUSION: EGFRvIII could induce autophagy, and CH12 treatment could improve autophagy activity in EGFRvIII-positive liver cancer cells. The combination of CH12 with an autophagy inhibitor or siRNA against key proteins in the autophagy pathway displayed more significant efficacy on EGFRvIII-positive tumor cells than monotherapy, and induced cell apoptosis.

Autophagy suppresses the proliferation of renal carcinoma cell.

OBJECTIVE: To investigate the effetcs of autophagy on the proliferation of renal carcinoma (RCCs). MATERIALS AND METHODS: Authophagy related protein 7 (Atg7)-overexpressing and knockdown RCC cell lines were established using lentiviral transfection and shRNA interference, respectively. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) was used to determine the Cell growth rate, and western blot was used to determine the expression of protein. In order to establish xenograft animal models, stable transfected cells were injected into nude mice. After that those mice were used to detect the effect of autophagy on the growth of RCC in vivo. RESULTS: Atg7 overexpression could up-regulate the level of LC3II in RCC cell lines, while Atg7-knockdown suppressed the expression of light chain 3 II (LC3II) in RCC cell lines. Atg7-overexpression cells exhibited a decreased growth profile, while suppressing the expression of Atg7 could accelerate the growth of RCC formed tumors. CONCLUSIONS: Our data indicated that autophagy could suppress the growth of RCC cells in vivo and in vitro, and autophagy appeared to be a new therapeutic target for treating advanced renal cell carcinoma.

IL-17A induces autophagy and promotes microglial neuroinflammation through ATG5 and ATG7 in intracerebral hemorrhage.

Microglial inflammation plays a vital role in intracerebral hemorrhage (ICH)-induced secondary brain injury. IL-17A has been identified to promote microglia activation, but the role in the pathology following ICH remains unclear. Autophagy is involved in modulation of cell metabolism, cell survival, and immune response. However, the role of IL-17A in autophagy following ICH has not been well defined. In this study, we assessed the role of IL-17A in microglial autophagic activity following ICH. The microglia were treated with IL-17A, and then autophagy and inflammation were detected. In addition, RNA interference in essential autophagy genes (ATG5 and ATG7) was also utilized to analyze microglial autophagy in vitro. Furthermore, ICH mice were made by injection of autologous blood model in vivo. And the IL-17A-neutralizing antibody was utilized to assess the neurological scores and brain edema. These data demonstrated that IL-17A promoted microglial autophagy and microglial inflammation. The suppression of autophagy using RNA interference in essential autophagy genes (ATG5 and ATG7) decreased microglial autophagy and inflammation. Moreover, IL-17A Ab significantly reduced brain water content and improved neurological function of ICH mice. Taken together, these data demonstrated that IL-17A promoted microglial autophagy and microglial inflammation, and IL-17A-mediated activation of autophagy might represent novel clues in ICH therapy.

Nutritional shortage augments cisplatin-effects on murine melanoma cells.

Melanoma incidence increases every year worldwide and is responsible for 80% of skin cancer deaths. Due to its metastatic potential and resistance to almost any treatments such as chemo, radio, immune and targeted-therapy, the patients still have a poor prognosis, especially at metastatic stage. Considering that, it is crucial to find new therapeutic approaches to overcome melanoma resistance. Here we investigated the effect of cisplatin (CDDP), one of the chemotherapeutic agents used for melanoma treatment, in association with nutritional deprivation in murine melanoma cell lines. Cell death and autophagy were evaluated after the treatment with cisplatin, nutritional deprivation and its association using an in vitro model of murine melanocytes malignant transformation to metastatic melanoma. Our results showed that nutritional deprivation augmented cell death induced by cisplatin in melanoma cells, especially at the metastatic subtype, with slight effects on melanocytes. Mechanistic studies revealed that although autophagy was present at high levels in basal conditions in melanoma cells, was not essential for cell death process that involved mitochondrial damage, reactive oxygen species production and possible glycolysis inhibition. In conclusion, nutritional shortage in combination with chemotherapeutic drugs as cisplatin can be a valuable new therapeutic strategy to overcome melanoma resistance.