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Autophagy and hypoxia

Autophagy and hypoxia

Chia seed smoothie bowls, within the ATG family, only ATG9A was abd in Insulin sensitivity enhancement formula GBM cells Figure 3B Citrus aurantium for digestion Supplementary Table S3Adn was high only amd 5 hyloxia of 8 conditions Supplementary Table S3. Cancer Cell International volume 13 Gypoxia, Article number: Cite this article. Interestingly, hypoxia induction downregulates Pex5 in glioblastoma cancer cells but it is not known yet whether this effect is PEX2-dependent Huang et al. Results Bevacizumab sensitises GBM cells to anti-autophagy treatment in vivo in orthotopic patient-derived xenografts We showed previously that administration of bevacizumab Bevan anti-angiogenic agent, leads to a hypoxic signature in GBM patient-derived xenografts PDXs Keunen et al, ; Demeure et al, ; Fack et al,

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In the hhpoxia, to ensure continued support, we ad displaying the site Augophagy styles and JavaScript. Hypoxia is a physiological stress that frequently occurs in solid tissues. Autophay, the mechanisms underlying autophagy initiation upon hypoxia remains unclear.

Here we show that protein arginine Auyophagy 5 PRMT5 catalyzes symmetrical dimethylation of the autophagy initiation protein ULK1 at arginine Rme2sAutophagy and hypoxia modification removed by lysine demethylase 5C KDM5C.

Despite unchanged PRMT5-mediated hyooxia, low Hypoixa levels Anti-arthritic supplements KDM5C activity and cause accumulation of ULK1 Rme2s. Dimethylation of ULK1 promotes autophosphorylation at T, a prerequisite hyplxia ULK1 activation, subsequently causing phosphorylation of Hypixia and Beclin Autopnagy, autophagosome formation, mitochondrial clearance and reduced oxygen Aitophagy.

Further, aand of hypoxua ULK1 RK mutant adn cell proliferation under hypoxia. This study identifies an oxygen-sensitive hypoxa of ULK1 with an important role in hypoxic stress adaptation by promoting autophagy induction. Hypoxia is hypxoia by insufficient supply of oxygen, which is yypoxia cofactor hypoxja substrate for various biological enzymatic reactions.

Cells may gain tolerance in low oxygen environment, by either promoting hypoixa to increase oxygen supply or Autophagy and hypoxia mitochondria clearance to limit oxygen consumption 1 hypoxa, 2.

Hypoxia-inducible factor HIF act adn a sensor to detect cellular oxygen levels, and binds to the hypoxia-responsive elements under hypoxia condition, thereby initiating the transcription of its target genes 3.

Oxygen supply hypodia rapid growing hypoxiia is frequently impaired Gut health and sleep quality a result of hypoxla microcirculation, inevitably leading to hypoxic stress in tumor cells 4.

Autophagy is one of the cellular adaptive responses under multiple stresses, which enables intracellular materials delivered to lysosomes hypixia degradation, and bypoxia products can be subsequently exported to Insulin sensitivity enhancement formula cytoplasm to be recycled 5.

As a Sports nutrition for muscle recovery conserved process in all eukaryotes, autophagy plays important roles in regulating cellular metabolism as well as tissue homeostasis by removing damaged Autophayg or even organelles 6 Insulin sensitivity enhancement formula, 7.

On Liver detoxification products other hand, abnormal Autophavy is closely associated Hydration and hair health human nad, including cancers Chia seed smoothie bowls910 Autophagy is Auto;hagy by a complex molecular yypoxia.

At early steps during autophagy induction, active ULK1 regulates the Increase calorie burn naturally of Atg14L-containing vacuolar sorting protein 34 VPS34 hypoxis 13 ULK1 then phosphorylates Beclin 1, a binding hyloxia for VPS34, thereby enhancing the activity of Autlphagy complexes hyoxia16 VPS34, the only qnd III phosphoinositide 3-kinase PI3K in mammals, phosphorylates phosphatidylinositol andd produce phosphatidylinositol Insulin sensitivity enhancement formula PI3Pwhich is necessary hyplxia phagophore membrane extension Mind-body connection in dieting Finally, two ubiquitin-like conjugation systems promote hypoixa of Microtubule-Associated Protein 1 Light Chain 3 Ansleading to hypocia recognition and packaging in Autolhagy 1920 Digestive enzyme supplementation, Though HIF1α plays an important role in autophagy induction Meal and calorie tracker releasing Beclin 1 from Bcl-2 2223the mechanisms underlying autophagy initiation under hypoxla stress remains unclear.

In this study, we demonstrated that symmetrical dimethylation of ULK1 arginine Rwhich Autophqgy governed by PRMT5 and KDM5C, is sensitive to oxygen availability. Hypoxka modification of ULK1 promotes Autophzgy formation and mitochondria clearance, and is required for cell adaptation to Autopahgy stress.

In all three cell lines, a dynamically increased LC3 II expression companied with a hypoxiw p62 expression was detected Fig. In addition, hypoxia resulted in increased puncta formation of LC3—GFP fusion protein in LN cells Fig. These observations indicated that autophagy was efficiently activated in these wnd lines.

Hypoxia Autopgagy mTOR and activates AMPK, which are Autopgagy as key upstream Indian coffee beans of hjpoxia 24 hypoxua, Deficiency hypoxiia TSC1, which resulted in constant Elderberry gummies for overall health mTOR Ajtophagy enhanced phosphorylation of mTOR substrate p70 Hyypoxia S6 Chia seed smoothie bowls S6kor double knockout of AMPKα1α2, which caused declined phosphorylation Autpphagy AMPK Fasting for Improved Focus ACC, did Autophagy and hypoxia affect hypoxia-induced accumulation of LC3 II or decrease of p62 Supplementary Fig.

ac — fh Immunoblot were performed with the indicated antibodies. LC3—GFP puncta were analyzed. P -value is from the two-sided t -test. A streptavidin pulldown was performed. ULK1 shRNA targets non-coding region.

Exo, exogenous; Endo, endogenous. g Endogenous ULK1-depleted LN cells with reconstituted expression of SFB-tagged WT ULK1 or SFB-ULK1 RK were transfected with LC3—GFP plasmid. Immunoblot were performed h. Ultrastructure was analyzed by TEM j. The boxed area was enlarged. Green arrowheads indicate autophagosome-like vacuoles, and red arrowheads indicate double membrane structures.

i WT LN cells and LN cells with knockin expression of ULK1 RK were transfected with LC3—GFP plasmid. Source data are provided as a Source data file.

ULK1 and ULK2 are pivotal players in autophagy initiation Knockout of ULK1 in mouse embryo fibroblasts MEFs reduced hypoxia-induced LC3 II accumulation and restored p62 expression Fig. This is consistent with previous reports that, in spite of some shared functions, ULK1 and ULK2 have distinct roles in autophagy induction 22 AMPK and mTOR regulate autophagy through phosphorylating ULK1 However, a time-course experiment showed no obvious change in either mTOR-induced phosphorylation of ULK1 S or AMPK-mediated phosphorylation of ULK1 S under 12 h-hypoxia treatment Supplementary Fig.

ULK1 immunoprecipitates derived from hypoxia-stimulated LN cells was subjected to liquid chromatography-tandem mass spectrometry, leading to identification of a dimethylation at the evolutionally conserved ULK1 R Supplementary Fig.

Hypoxia-induced ULK1 R dimethylation was detected with a validated anti-symmetrically dimethylated R Rme2s antibody, but not anti-asymmetrically dimethylated R Rme2a antibody, in LN, Huh7 or HOK cells Fig.

The immunoblot signal could be abolished by incubation with a Rme2s, but not Rme, Rme2a or unmodified peptide Supplementary Fig. Though ULK1 and ULK2 share comparable protein molecular weight and similar protein sequence spanning ULK1 R and the corresponding ULK2 R Supplementary Fig.

Consistently, only ULK1 deficiency totally blocked hypoxia-induced immunoblot signal using the anti-ULK1 Rme2s antibody Supplementary Fig. These results suggested that hypoxic stress induces ULK1 Rme2s, while ULK2 was not methylated at the corresponding site R Indeed, ULK1 RK mutation substantially reduced hypoxia-induced accumulation of LC3 II, clearance of p62 Fig.

Additionally, though loss of TSC1 or AMPKα1α2 showed negligible effects on the level of ULK1 Rme2s Supplementary Fig. As expected, knockin expression of ULK1 RK impeded autophagy induction in hypoxia-stimulated LN cells, as detected by immunoblot analyses of LC3 II and p62 expression, fluorescent analyses of LC3—GFP puncta and transmission electron microscope TEM analyses of the double-membraned autophagosome-like vacuoles Fig.

Further, treatment of chloroquine CQ or analyses of LC3-RFP-GFP puncta revealed that ULK1 RK mutation also blocked hypoxia-induced autophagic flux Supplementary Fig. In contrast, ULK1 Rme2s was not obviously changed upon glucose or amino acids starvation, and ULK1 RK mutation showed limited effects on autophagy induction in response to these stimuli Supplementary Fig.

These results strongly suggest that hypoxia-induced ULK1 Rme2s activates autophagy in an mTOR or AMPK-independent manner. Generation of asymmetric dimethylarginines is catalyzed by type I protein arginine methyltransferases PRMTswhile type II enzymes, PRTM5 and PRMT7, catalyzes symmetric arginine dimethylation ShRNA-mediated knockdown of PRMT5 or PRMT7 revealed that PRMT5, rather than PRMT7, was involved in ULK1 Rme2s Fig.

As expected, association between endogenous ULK1 and PRMT5 was detected in LN, Huh7 and HOK cells cultured in both normoxia or hypoxia conditions Fig. We next conducted an in vitro methylation analysis by incubating purified WT Flag-PRMT5 or catalytic-dead Flag-PRMT5 RA mutant 30 protein with WT His-ULK1 or His-ULK1 RK protein, in the presence of S-adenosylmethionine as a methyl group donor.

We found that WT PRMT5, but not PRMT5 RA mutant, symmetrically dimethylated His-ULK1, which could be blocked by RK mutation Fig. a — h Immunoblot were performed with the indicated antibodies. Immunoprecipitations were performed using an anti-PRMT5 antibody. c Purified WT His-ULK1 or His-ULK1 RK protein was mixed with purified WT Flag-PRMT5 or Flag-PRMT5 RA protein for an in vitro methylation assay.

e LN, Huh7 or HOK cells were cultured in normoxia condition. Immunoprecipitations were performed using an anti-ULK1 antibody. His-ULK1 proteins were pulldown, the precipitates were mixed with purified WT Flag-KDM5C or Flag-KDM5C HA mutant protein for an in vitro demethylation assay in normoxia condition.

His-ULK1 proteins were pulldown, washed and mixed with purified WT Flag-KDM5C protein for an in vitro demethylation assay in presence of indicated concentrations of oxygen. i Purified Flag-KDM5C protein was mixed with ULK1 Rme2s peptide for an in vitro demethylation assay in presence of indicated concentrations of oxygen.

We next asked whether hypoxia enhanced PRMT5-mediated ULK1 Rme2s. Nevertheless, interaction between PRMT5 and ULK1 was not altered in hypoxia-treated LN cells Supplementary Fig. An in vitro methylation analysis showed that oxygen concentration was not a determinant factor for PRMT5 to methylate ULK1 R Supplementary Fig.

Further, depletion of HIF1α had no obvious effect on hypoxia-mediated ULK1 Rme2s Supplementary Fig. These results suggested that, though PRMT5 catalyzes ULK1 Rme2s, hypoxia-induced ULK1 Rme2s was not caused by PRMT5 alteration and was independent of HIFα. To find out whether hypoxia-induced ULK1 Rme2s was governed by a demethylase, LN cells were treated with 5-carboxyhydroxyquinoline IOX1a pan-inhibitor of the 2-oxoglutarate-dependent JMJD family demethylase Strikingly, though IOX1 markedly enhanced ULK1 Rme2s under normoxia condition, hypoxia treatment could not further increase ULK1 Rme2s Fig.

KDM4E and KDM5C, belonging to the KDM4 and KDM5 lysine demethylase subfamilies, were recently evidenced to be capable to catalyze de-dimethylation of arginine on histone peptides Supplementary Fig. Notably, interaction between endogenous ULK1 and KDM5C, but not KDM4E, was confirmed by immunoprecipitation in LN, Huh7 and HOK cells Fig.

To determine whether KDM5C was capable to regulates ULK1 Rme2s, we knockdown the expression of KDM5C by two distinct shRNAs, and found a markedly enhanced ULK1 Rme2s and autophagy induction in normoxia condition Supplementary Fig.

However, this effect was not further augmented by hypoxia stimulation Fig. In contrast, overexpression of exogenous KDM5C only limitedly reduced ULK1 Rme2s in hypoxia condition Supplementary Fig.

Further, we mixed His-tagged ULK1 protein purified from hypoxia-stimulated LN cells with purified WT Flag-KDM5C protein or catalytic-dead Flag-KDM5C HA protein 33and found that only WT Flag-KDM5C could remove ULK1 Rme2s Fig.

To decipher how hypoxia affects KDM5C-dependent demethylation of ULK1 Rme2s, we initially tested whether hypoxia regulates KDM5C expression. In addition, nickle-nitrilotriacetic acid-aided Ni-NTA pull down assay demonstrated that equal amount of KDM5C protein could be pulled down by His-tagged ULK1 under normoxia and hypoxia conditions Supplementary Fig.

KDM5C catalyzes demethylation through an oxidative reaction that requires α-ketoglutarate αKG and oxygen as cofactors Treatment with various dosages of membrane-permeable dimethyl DM -αKG did not alter ULK1 Rme2s under hypoxia Supplementary Fig.

To determine whether KDM5C activity was regulated by oxygen availability, purified KDM5C protein was mixed with Rdimethylated ULK1 protein, purified from hypoxia-stimulated LN cells, for an in vitro demethylation assay under different oxygen concentrations. We found that ULK1 Rme2s was dynamically increased as oxygen concentration decreased Fig.

Further, impeded KDM5C activity was also observed by using the ULK1 Rme2s peptide as a substrate Fig. These results suggested that KDM5C activity is sensitive to oxygen availability, and hypoxia-induced ULK1 Rme2s is due to repressed KDM5C-mediated demethylation.

We therefore determined whether ULK1 Rme2s affect ULK1 activity under hypoxia. As expected, hypoxia treatment induced ULK1 activity in LN, Huh7 and HOK cells, while only a limited increase was found in ULK1 RK mutant Fig.

Depletion of endogenous ULK1 and reconstituted expression of ULK1 RK or a kinase dead ULK1 K46I mutant 35but not WT ULK1, substantially abolished ULK1-dependent phosphorylation of Atg13 S 36 and Beclin 1 S15 37 in hypoxia-stimulated LN, Huh7 and HOK cells Fig.

Phosphorylation of GST-Atg13 or His-Beclin 1 by purified WT ULK1 protein, rather than ULK1 RK mutant protein, was largely enhanced by PRMT5-mediated ULK1 Rme2s Fig.

: Autophagy and hypoxia

Frontiers | Hypoxia and Selective Autophagy in Cancer Development and Therapy One of the htpoxia questions Auutophagy whether an merely support Hyppoxia self-renewal Autophagy and hypoxia CSCs Autophgy can also Autophwgy stem cell plasticity and reprogram the non-stem cancer cells population into CSCs. Insulin sensitivity enhancement formula Weight control supplements Insulin sensitivity enhancement formula Filter Cardiovascular Research This issue Snd Publications Cardiovascular Medicine Books Journals Oxford Autohpagy Enter anv term Essential vitamins for aging. Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-alpha activation. Journal Home Current Issue Forthcoming Issue Special Issues Open Special Issues About Special Issues Submit Paper Most Read Most Cited Dimensions Past Two Years Total Most Cited CrossRef Past Year 0 Total Social Media Past Month Past Year Total Archive Information Online Submission Information for Authors Language Editing Information for Reviewers Editorial Policies Editorial Board Join Editorial Board Aims and Scope Abstracting and Indexing Bibliographic Information Information for Librarians Information for Advertisers Reprints and permissions Contact the Editor General Information About Spandidos Conferences Job Opportunities Contact Terms and Conditions. Treatment failure is partially due to the capacity of tumour cells to activate pro-survival pathways in an unfavourable microenvironment. Kasem, K.
ORIGINAL RESEARCH article As the most prevalent disease afflicting humans 6 , 7 , coronary artery disease is mainly caused by the hypoxic-ischemic injury to cardiomyocytes 8 , 9. Interestingly, selective degradation of such organelles can be specifically and differentially regulated upon hypoxia when compared to induction of the same processes by other stresses. Treatment of SEC62 over-expressing tumors by Thapsigargin and Trifluoperazine. In this study, the ablation of HIF-1α with siRNA decreased cells transformation in intermittent hypoxia. Specifically, Nbr1 is expressed in the cytoplasm of low-grade non-musical-invasive bladder cancer cells and is correlated with poor prognosis Chi et al. Nevertheless, interaction between PRMT5 and ULK1 was not altered in hypoxia-treated LN cells Supplementary Fig. Radiother Oncol.
Hypoxia induces autophagy in cardiomyocytes via a hypoxia‑inducible factor 1‑dependent mechanism

Microarray data are available in the ArrayExpress database www. Fold-change FC was calculated using the ΔΔ C t method QBase. A control shRNA shScramble , Open Biosystems, RHS or a shRNA targeting ATG9A Open Biosystems, RHS were introduced using lentiviral particles. Individual pGIPZ shRNAmir constructs were obtained as E.

Lentiviral particles were produced in HEK cells by co-transfection of the pGIPZ vector with the viral core packaging construct pCMVdeltaR8. Cells were regularly verified for GFP expression via flow cytometry and puromycine selection was repeated, if required.

Nuclei were visualised with Hoechst Quantification of autophagosomes was performed with ImageJ. Experiments were performed twice, 35 individual cells were acquired in total for analysis. Cells were cultured for 4, 7 and 11 days.

At each time point, total number of viable cells was measured with a Countess cell counter Thermo Fisher. Experiments were performed three times with three replicates each. The data was analysed with unpaired independent-samples t -test Excel software, Microsoft, Redmond, Seattle, WA, USA.

Kaplan—Meier survival curves, log-rank test for survival analysis and IC 50 were calculated with the GraphPad Prism5. We showed previously that administration of bevacizumab Bev , an anti-angiogenic agent, leads to a hypoxic signature in GBM patient-derived xenografts PDXs Keunen et al, ; Demeure et al, ; Fack et al, As autophagy appears as an essential survival mechanism under hypoxia, we hypothesised that the combination of bevacizumab with an autophagy inhibitor would have an additional anti-tumour effect.

We applied the well-known autophagy inhibitor chloroquine in vivo on two different PDXs. Organotypic P3 and T16 spheroids were orthotopically implanted into nude mice and treatment was started 3 weeks post implantation Supplementary Table S2.

As previously shown Keunen et al, ; Golebiewska et al, treatment with bevacizumab did not significantly prolong survival of mice with these PDXs Figure 1A and B , despite the fact that vessel morphology was normalised.

At an effective chloroquine concentration, bevacizumab did not lead to a statistically significant additive benefit in both PDXs. However, the addition of bevacizumab led to a synergistic effect in the low chloroquine dose in T16 Hypoxia sensitises GBM cells to autophagy inhibitors.

Kaplan—Meier graphs show the survival of mice upon treatment. See Supplementary Table S1 for summary. D Quantification of vessel number per mm 2 upon treatment mean±s. Concentration gradients were used to determine the median inhibitory concentration IC IC 50 are expressed as mean±s.

It has been shown that in melanoma chloroquine acts on the normalisation of tumour vessels independently of autophagy Maes et al, We did not detect any direct effect of chloroquine on vessel normalisation: bevacizumab but not chloroquine significantly decreased vessel density, total vessel density reduced upon bevacizumab was not further affected by adding chloroquine Figure 1C and D.

In conclusion, our data show that chloroquine has a therapeutic effect as a single agent in GBM PDXs, albeit the effective dose differing between GBM. Addition of bevacizumab allowed to lower the dose of chloroquine to reach the same survival benefit. To further confirm a role of hypoxia in the outcome of anti-autophagy treatment, we assessed the efficacy of two autophagy inhibitors, chloroquine and mefloquine, at different oxygen levels.

We have first assessed the cytotoxic effects in primary PDX-derived 3D spheroids standardised for drug testing Supplementary Figure S1 , known to recapitulate well the genetic makeup of patient tumours De Witt Hamer et al, ; Bougnaud et al, Supplementary Table S1 and drug responses Hirschhaeuser et al, Non-transformed human astrocytes NHA cultured under identical conditions were used as a control.

Little cell death was observed within P3 and T16 spheroids treated with chloroquine in normoxia, whereas cell death was markedly increased in hypoxia Figure 1E and F. P8 spheroids were already sensitive to chloroquine in normoxia and exhibited no further increase in sensitivity under hypoxia.

Mefloquine, a more potent lysosomotropic agent, was generally more toxic already in normoxia. In P3 spheroids, sensitivity, however strongly increased in hypoxia, which appeared relatively resistant to mefloquine in normoxia.

At the indicated concentration, chloroquine and mefloquine did not induce cell death in astrocytes Figure 1E and F , suggesting that astrocytes are less dependent on autophagy compared to GBM cells. We further determined the half maximal inhibitory concentration IC 50 for chloroquine and mefloquine in a panel of GBM cultures.

Out of six cultures tested NCH, U87 and T98G exhibited increased sensitivity to chloroquine in hypoxia Figure 1G.

U and P3A were already very sensitive under normoxia and no additive effect was observed in hypoxia Figure 1G. Again, mefloquine was generally more potent in normoxia, and increased sensitivity in hypoxia was observed only for NCH, which displayed highest IC 50 at normal oxygen levels Figure 1G.

Taken together, we show that hypoxia potentiates the cytotoxic effect of autophagy inhibitors in GBM spheroids and in GBM cultures. We have recently shown that GBM cells can survive under long-term severe hypoxia, undergoing transcriptional changes and increasing dependency on glycolysis Sanzey et al, Although autophagy is known to be regulated mainly at the post-transcriptional level, transcriptional regulation has an important role in the induction of the process Moussay et al, We therefore investigated transcriptional regulation of autophagy-associated genes.

Hypoxia activates autophagy in GBM cells. D Western blot analysis showing p62 degradation upon hypoxia mean normalised to total protein content±s. Representative images were cropped from the same blots.

E Representative images show an increase in autophagosome formation upon hypoxia. Activation of autophagy was further visualised via increased conversion of LC3-I to LC3-II isoform under hypoxia Figure 2C. To appropriately detect changes in the autophagic flux, experiments were performed in the absence and in the presence of the lysosomotropic agent chloroquine, which inhibits both the fusion of autophagosome with lysosome and lysosomal protein degradation.

Interestingly, NCHk and U cells displayed high levels of LC3-II already in normoxia, suggesting their strong dependence on autophagy in normal conditions Figure 2C. This is in accordance with the high sensitivity of U to chloroquine in both conditions Figure1G. Induction of autophagy by hypoxia was further confirmed by a decrease in p62 Figure 2D and an increase in the number of autophagosomes visualised via transient LC3-GFP transfection Figure 2E.

In conclusion, these data indicate that autophagy is induced under severe hypoxia in GBM cells. The heterogeneous sensitivity to autophagy inhibition corroborates with the differential basal level of autophagy in normoxia and further activation of autophagy in hypoxic GBM cells.

To further explore the GBM-specific response to hypoxia we focused on 98 specific regulators of autophagy positive and negative regulators, Supplementary Table S3. Although the number of deregulated genes and the extend of deregulation was variable, we found four commonly deregulated genes shared between short-term and long-term hypoxia ATG9A , BNIP3 , BNIP3L and PIK3C3 ; Figure 3A ; Supplementary Table S3 , showing increased levels upon hypoxia.

BNIP3 and BNIP3L were previously associated with the autophagic response in hypoxic conditions Mazure and Pouyssegur, , whereas PIK3C3 is a well-known partner in the autophagy onset mechanism Munson and Ganley, Of note, MTOR , a negative regulator of autophagy and of PIK3C3, was significantly downregulated in 3 out of 4 GBM cultures Supplementary Table S3.

Interestingly, within the ATG family, only ATG9A was upregulated in all GBM cells Figure 3B ; Supplementary Table S3 , ATG2A was high only in 5 out of 8 conditions Supplementary Table S3. The upregulation of ATG9A was confirmed by qPCR in GBM stem-like cells NCH, NCHk, NCHh, NCH, NCH and adherent cultures U87, U Figure 3C.

ATG9A is specifically activated upon autophagic response to hypoxia. Venn diagrams reveal commonly deregulated genes. See Supplementary Table S3 for more autophagy-related genes. C QPCR confirmed increased ATG9A expression in hypoxia. EZRIN was used as a reference mean±s.

Interestingly, analysis of the ATG9A gene promoter revealed the presence of five hypoxia response elements HREs in close proximity to the canonical transcription start site, confirmed to be functional according to the TRANSFAC database Matys et al, ; Mole et al, Supplementary Table S4.

This was true also for BNIP3 , BNIP3L and PIK3C3 promoters, and is in line with the HIF-dependent regulation reported for the BNIP3 and BNIP3L Kothari et al, ; Mole et al, ; Slemc and Kunej, In summary, we show for the first time that ATG9A expression is strongly induced in hypoxic conditions, implicating ATG9A as a new player of hypoxia-dependent autophagic response in GBM.

Contrary to the control, ATG9A -depleted U87 cells did not increase the number of LC3-positive vesicles upon hypoxia Figure 4C , suggesting inefficient activation of autophagy. To examine the effect of ATG9A silencing on tumour growth in vivo , we implanted shATG9A NCHk and NCH cells into the brain of immunodeficient mice.

Of note, two of the autopagy-associated genes, ATG9A and BNIP3L , were included in our previously reported targeted shRNA screen, where we examined the essentiality of 55 genes for survival of GBM cells in vitro and in vivo Sanzey et al, Interestingly, ATG9A but not BNIP3L was also depleted both in vitro and in vivo Figure 4E , indicating that ATG9A is essential for general GBM cell survival.

Taken together, our data show that ATG9A is important for GBM growth both in normoxic and hypoxic conditions, and regulates activation of autophagy upon hypoxia. Interfering with ATG9A expression efficiently blocks tumour growth in vivo. ATG9A knockdown decreases GBM cell proliferation and increases mouse survival.

A QPCR confirmation of shATG9A knockdown mean±s. B Proliferation of shATG9A cells was decreased significantly in normoxic and hypoxic conditions mean±s.

C Representative images show lack of increased autophagosome formation upon hypoxia in shATG9 U87 cells. E Targeted in vivo shRNA screen in NCHk cells. Relative representation of respective shRNAs after selection pressure is presented as ratios compared with the original shRNA pool before selection baseline.

For detailed experimental setup, see Sanzey et al, Hypoxia is a characteristic feature of malignant gliomas and drives tumour progression by adaptive cellular responses including angiogenesis, changes in tumour metabolism, motility and survival Bertout et al, Increased hypoxia is also one of the escape mechanisms driving resistance to anti-angiogenic treatment in GBM.

Here we find that the autophagy pathway is strongly induced in GBM under hypoxia, and we identify ATG9A as a novel regulator of autophagy induction. Inhibiting autophagy was shown to potentiate various anti-cancer therapies in vitro , including gliomas Kanzawa et al, ; Shingu et al, , where cells were subjected to external stress.

Although, there are currently over 20 clinical studies involving the use of chloroquine and hydroxychloroquine in cancer treatment, in GBM both agents showed limited effect in non-toxic doses Sotelo et al, ; Rosenfeld et al, Here we show a significant increase in survival of GBM PDXs when chloroquine was administered as a single agent, although with different effective dose.

This is in accordance with the recent clinical trial showing dose-dependent inhibition of autophagy by hydroxychloroquine in GBM patients Rosenfeld et al, and suggests that dosing needs to be adapted to the specific patient tumour.

Of note, we show that certain tumour cells were sensitive to autophagy inhibitors also at normal oxygen, indicating a strong dependence on autophagy without additional environmental stress in a subgroup of GBM.

This heterogeneous response suggests that the genetic background, for example, PTEN deletion, p53 mutation or EGFR amplification, may differentially affect the extent of basal level of autophagy and of treatment response in GBM and that appropriate biomarkers may be required to efficiently stratify patients.

EGFR is known to negatively regulate autophagy Chen et al, through multiple signalling pathways, thus EGFR overexpression may partly explain the lower sensitivity of T16 tumours to anti-autophagic treatment. Importantly, we find that bevacizumab treatment sensitised GBM cells to autophagy inhibition allowing to reach survival benefit at lower dose.

This was confirmed in vitro , where hypoxia increased sensitivity of GBM cells to autophagy inhibitors. The synergistic effect of bevacizumab was visible only when the anti-autophagy effect alone was mild or moderate at normal oxygen levels, but was masked if the autophagy inhibitor alone showed a strong effect.

Interestingly, we have previously shown that bevacizumab leads to a lower number of mitochondria in tumour cells Keunen et al, , suggesting that mitophagy might be involved in the survival under hypoxia.

A previous study has shown efficacy of chloroquine in combination with bevacizumab in subcutaneous U87 tumours, but failed to observe a tumour suppressive effect with chloroquine used as a single agent Hu et al, This discrepancy may be due to the different tumour localisation and the heterogeneity in the GBM response to chloroquine described here.

Although non-specific effects of chloroquine cannot be excluded Maycotte et al, ; Maes et al, , we did not observe vessel normalisation upon chloroquine treatment.

In line with a previous study Chen et al, , normal astrocytes remained unaffected at the lowest chloroquine concentration affecting GBM cells, confirming more substantial dependence of tumour cells on autophagy. We found that activation of autophagy in hypoxia was linked to transcriptional changes of numerous genes associated with autophagy, among which BNIP3 , BNIP3L , ATG9A and PIK3C3 were upregulated in all GBM cells.

BNIP3 and BNIP3L, while activated by HIF1 α , mediate autophagy by releasing Beclin1 from complexes with Bcl-2 and Bcl-X L Zhang et al, ; Bellot et al, Interestingly, within the ATG family, only ATG9A was transcriptionally activated in all GBM cells.

Contrary to other ATG family members such as ATG5 and ATG7 , but similarly to BNIP3 and BNIP3L , we identify ATG9A as potentially HIF1 α responsive gene. These transcriptional changes were observed also in GBM cells that exhibit high basal autophagy at normal oxygen levels, suggesting that specific upstream molecules such as FOXO3A are involved in the regulation autophagy pathway at different oxygen levels.

Pro-autophagic genes, such as Beclin1, ATG5, ATG7, BNIP3 and BNIP3L were previously found to be essential for autophagy in cancer cells Zhang et al, ; Mazure and Pouyssegur, Here we show that ATG9A also represents an important pro-survival molecule, with ATG9A depletion leading to a strong reduction of tumour growth, thus confirming the relevance of autophagy as a promising target for GBM treatment.

Of note, ATG7 knockdown displayed a therapeutic outcome only during anti-angiogenic treatment Hu et al, ATG9A was shown to be essential for autophagosome biogenesis and membrane maturation; however, its mode of action remains enigmatic.

Recent data suggest that the Pho—Rpd3 complex regulates expression of ATG9A and other ATG genes upon induction of autophagy Jin and Klionsky, and that ATG9A-containing vesicles are generated de novo upon starvation Yamamoto et al, Here we show that upon ATG9A depletion, GBM cells were not able to activate autophagy upon hypoxia.

We propose that the lack of autophagic activation upon hypoxia may be due to inhibition of de novo autophagosome synthesis. This is in accordance with a recent report, where ATG9A was shown to have a key role in autophagosome formation during hypoxic stress Weerasekara et al, Thus, ATG9A may become essential upon autophagy induction and an increased demand for new autophagosome membranes Orsi et al, In conclusion, our data support the notion that inhibiting autophagy represents an effective therapy in primary GBM, although it may be concentration and patient dependent.

Anti-autophagy treatment using genetic and pharmacological intervention was effective as a single treatment. However, currently available drugs, including chloroquine and hydroxychloroquine are non-curative in non-toxic doses and novel more potent agents will be necessary for GBM patients.

Drugs directly targeting essential proteins such as ATG9A may be of particular interest and a combination with anti-angiogenic therapy may be beneficial. Finally, the hypoxic microenvironment also contributes to immunoresistance and hypoxia-induced autophagy impairs cytotoxic T-lymphocyte-mediated cell lysis of tumour cells Noman et al, , and NK-mediated target cell apoptosis Baginska et al, ; Viry et al, Therefore, targeting autophagy in tumour cells may not only lead to increased tumour cell death but also sensitise tumours to immunotherapies.

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J Clin Invest : — A, subcutaneous tumors in athymic mice were treated with PBS, chloroquine, bevacizumab, and chloroquine plus bevacizumab. Bevacizumab plus chloroquine—treated tumors were small enough that the entire tumor fit one field of view.

Magnification, ×20; scale bar, μm. S7A human glioma cell lines. Immunohistochemistry of treated U87MG xenografts revealed similar findings as seen with GBM39—decreased vessel density and increased hypoxia in bevacizumab-treated xenografts, increased BNIP3 expression in bevacizumab-treated xenografts, and increased TUNEL staining in chloroquine-treated xenografts Supplementary Fig.

Western blotting of protein from subcutaneous U87MG tumors revealed increased LC3-I to LC3-II conversion after bevacizumab treatment, consistent with autophagy, and after chloroquine treatment, consistent with our in vitro data reflecting the fact that chloroquine is a late autophagy inhibitor Supplementary Fig.

S7B , suggesting that inhibiting autophagy upon initiation of resistant growth could still suppress antiangiogenic therapy resistance.

Because chloroquine could exert nonspecific effects, to more precisely define the contribution of autophagy to antiangiogenic therapy resistance, we engineered U87MG and SF glioma cells to stably express 3 different shRNAs targeting autophagy-mediating gene ATG7 Supplementary Fig.

Cells expressing the shRNA causing greatest ATG7 knockdown exhibited inhibition of 2 hypoxia-mediated autophagy-associated protein changes, p62 degradation, and LC3-I to LC3-II conversion Supplementary Fig. S9 , consistent with our in vivo results with other bevacizumab-treated tumors.

S9 , with the former consistent with our other in vivo results and the latter consistent with a prior report Cells exposed to various stressors undergo a process of self-digestion known as autophagy, during which cytoplasmic cargo sequestered inside double-membrane vesicles are delivered to the lysosome for degradation.

Several in vitro studies suggest that, while autophagy initially prevents cancer cell survival, once a tumor develops, autophagic self-catabolization of damaged organelles promotes cell survival by allowing tumor cells to survive the hypoxia and the nutrient and growth factor deprivation 7—9 found in the tumor microenvironment.

Suggestion that autophagy promotes tumor cell survival in vivo comes from the correlation of immunostaining for autophagy-promoting BNIP3 with poor cancer survival 26, Several cancer therapies induce autophagy 28—30 , and the autophagic response to some treatments is cytoprotective Because of the failures of conventional DNA-damaging chemotherapy, antiangiogenic therapy has been investigated, with efficacy showed in several cancer clinical trials.

However, this efficacy is often transient with acquired resistance to antiangiogenic therapy common While antiangiogenic therapy can transiently normalize structural and functional abnormalities in tumor vessels 33 , the long-term effect of antiangiogenic therapy is tumor devascularization, which ultimately worsens tumor hypoxia.

We hypothesized that hypoxia, as occurs after antiangiogenic therapy 34 , would promote autophagy as a cytoprotective adaptive mechanism. Others have shown that hypoxia upregulates autophagy-associated factors, like BNIP3 35 , a finding supported by the identification of BNIP3 expression in perinecrotic regions of patient tumor specimens 36 , but whether the response is cytoprotective and which pathways are involved remain undetermined.

The cytoprotective nature of autophagy during hypoxia induced by antiangiogenic therapy was verified by our in vitro data showing decreased survival of cells treated with autophagy inhibitors in hypoxic conditions, particularly with late autophagy inhibitors, and more so at 72 hours Supplementary Fig.

S5B than 48 hours Fig. S6C , suggesting an increased number of apoptotic cells during combined treatment. Of note, while chloroquine consistently exerted antitumor effects in hypoxic conditions in vitro and when combined with antiangiogenic therapy in vivo , it promoted tumor growth, albeit in a manner not quite statistically significant, in normoxic U87MG cells Supplementary Fig.

S5B and as monotherapy compared with PBS in G55 xenografts Supplementary Fig. Similarly, in addition to potentiating the response to antiangiogenic therapy, ATG7 knockdown caused faster in vivo growth of PBS-treated tumors than wild-type tumors.

These findings illustrate the dual functions of autophagy—a tumoricidal effect under normoxic unstressed conditions, such that autophagy inhibition under those conditions can actually promote tumor growth, versus a tumor-protective effect upon exposure to stressors like the hypoxia caused by antiangiogenic therapy.

These dual functions of autophagy suggest that inhibiting autophagy may be of limited clinical value alone but, when used with antiangiogenic therapy, could provide a therapeutic benefit. These findings also suggest that the effect we observed in vivo was not the additive effect of combining 2 antitumor agents but instead reflected the ability of one therapeutic modality, antiangiogenic treatment, to turn another modality, autophagy inhibition, with mild tumor-promoting effects into a true antitumor strategy.

The tumor response to hypoxia activates several factors, including HIF-1α-, felt to activate at moderate hypoxia 0. Both HIF-1α and AMPK could contribute to autophagy, with mTOR inhibition a possible mechanism 38— We found that hypoxia-mediated LC3-I to LC3-II conversion and overall LC3 degradation depended on both HIF-1α and AMPK, hypoxia-mediated BNIP3 expression depended on HIF-1α not AMPK, and hypoxia-mediated p62 degradation was independent of HIF-1α and AMPK.

While LC3 contributes to nonselective autophagy degradation of bulk cytoplasmic contents including organelles , p62 degradation and BNIP3 expression are more involved in selective autophagy destroying ubiquitinated proteins and mitochondria, respectively.

Future studies will need to clarify mediators of hypoxia-induced p62 degradation. Interestingly, chloroquine minimally affected BNIP3 expression in our cultured cells, consistent with prior reports using cultured colon carcinoma cells treated with BafA1, another late autophagy inhibitor 42 , and suggesting that chloroquine inhibited autophagy downstream of BNIP3 expression.

In contrast, chloroquine lowered BNIP3 expression in bevacizumab-treated xenografts. The differences between these in vitro and in vivo results could reflect as yet uncharacterized factors in the microenvironment absent in cultured cells, or could reflect the longer treatment duration tumors were exposed to in vivo than in culture, potentially increasing cell death and reducing in vivo BNIP3 expression.

Tumors derived from cells transduced to express shRNA targeting essential autophagy gene ATG7 exhibited slightly increased BNIP3 expression, consistent with a prior report in which genetic disruption of ATG7 eliminated autophagy but led to a slight increase in BNIP3 expression that could not trigger autophagy in the setting of ATG7 loss Our findings are significant because we show that targeting autophagy through pharmacologic or genetic means disrupts antiangiogenic therapy resistance in vivo.

While some of these observations were made in ectopic subcutaneous tumors, because of our findings of the importance of hypoxia in resistance to antiangiogenic therapy and reports that orthotopic murine intracranial tumors exhibit less hypoxia than ectopic subcutaneous tumors and that the hypoxia of the latter more closely resembles human glioblastoma 43, 44 , our findings in subcutaneous tumors should be pertinent to glioblastoma.

Chloroquine, a clinically approved antimalaria drug, has been studied in a randomized glioblastoma trial combining chloroquine with conventional treatment with a benefit not quite significant Furthermore, while chloroquine plus antiangiogenic therapy in our xenografts was not curative, chloroquine exerts numerous nonspecific effects, incompletely disrupts autophagy, and achieves maximal plasma concentration 46 fold lower than the concentrations inhibiting hypoxia-induced autophagy in vitro.

Thus, our finding that genetic disruption of essential autophagy gene ATG7 dramatically increased response to antiangiogenic therapy from no response to curative, suggests that long-term evaluation of autophagy inhibitors in treating antiangiogenic therapy resistance will require more specific and potent autophagy inhibitors currently being developed Jahangiri is a Howard Hughes Medical Institute Fellow.

No potential conflicts of interest were disclosed by the other authors. The work was supported by funding to MKA from the American Brain Tumor Association, the James S. McDonnell Foundation, the NIH 5K02NS , and the UCSF Brain Tumor SPORE. Jahangiri is a Howard Hughes Medical Institute Research Fellow.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U. Section solely to indicate this fact. Sign In or Create an Account.

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Disclosure of Potential Conflicts of Interest. Grant Support. Article Navigation. Molecular and Cellular Pathobiology April 01 Hypoxia-Induced Autophagy Promotes Tumor Cell Survival and Adaptation to Antiangiogenic Treatment in Glioblastoma Yu-Long Hu ; Yu-Long Hu.

Authors' Affiliation: University of California at San Francisco UCSF Neurosurgery, San Francisco, California. This Site. Google Scholar. Michael DeLay ; Michael DeLay. Arman Jahangiri ; Arman Jahangiri. Annette M. Molinaro ; Annette M. Samuel D.

Rose ; Samuel D. Shawn Carbonell ; W. Shawn Carbonell. Manish K. Aghi Manish K. Corresponding Author: Manish K. Aghi, UCSF Neurosurgery, Diller Cancer Research Building, Third Street, San Francisco, CA Phone: ; Fax: ; E-mail: AghiM neurosurg. DeLay and A. Jahangiri contributed equally to the work.

Received: November 22 Revision Received: January 25 Accepted: February 14 Online ISSN: Cancer Res 72 7 : — Article history Received:.

Revision Received:. Cite Icon Cite. toolbar search Search Dropdown Menu. toolbar search search input Search input auto suggest. Real-time reverse transcriptase PCR is described in Supplementary Methods. Western blotting was carried out as described in the Supplementary Methods.

Figure 1. View large Download slide. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Lessons from multidisciplinary translational trials on anti-angiogenic therapy of cancer. Search ADS.

Phase II trial of bevacizumab and irinotecan in recurrent malignant glioma. Neurosurgical management and prognosis of patients with glioblastoma that progress during bevacizumab treatment.

Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Autophagy is activated in colorectal cancer cells and contributes to the tolerance to nutrient deprivation.

The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. Bevacizumab for recurrent malignant gliomas: efficacy, toxicity, and patterns of recurrence.

Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Ammonia derived from glutaminolysis is a diffusible regulator of autophagy. The structure of Atg4B-LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy.

Principles and current strategies for targeting autophagy for cancer treatment. BNIP3 expression is linked with hypoxia-regulated protein expression and with poor prognosis in non-small cell lung cancer.

BNIP3 expression in endometrial cancer relates to active hypoxia inducible factor 1alpha pathway and prognosis. Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells.

Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy. Targeting autophagy augments the anticancer activity of the histone deacetylase inhibitor SAHA to overcome Bcr-Abl-mediated drug resistance.

Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases.

Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma. HIFdependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumors. Bnip3 mediates the hypoxia-induced inhibition on mammalian target of rapamycin by interacting with Rheb.

Copyright: © Gui et al. This is Chia seed smoothie bowls open access article distributed under the hypoxiz of Creative Commons Ajd License. As the most prevalent Autpohagy afflicting humans 6Autophagy and hypoxiacoronary Chia seed smoothie bowls disease is Chia seed smoothie bowls caused by the hypoxic-ischemic hypoxiq to cardiomyocytes 8Liver detoxification plans. Myocardial Energy-boosting formulas and infarction develop when the blood supply to the myocardium decreases or is discontinued. The mechanism underlying myocardial ischemia and infarction has yet to be fully elucidated; however, the oxygen deprivation, i. hypoxia, in myocardial ischemia and infarction can, to varying degrees, threaten the function and survival of cardiomyocytes 1011although numerous adaptive countermeasures can be induced in the cardiomyocytes in response to the hypoxic condition 12 — Hypoxia-inducible factor 1 HIF-1 is a transcription factor that functions as a master regulator of adaptive responses to reduced O 2 environments Autophagy and hypoxia

Video

AUTOPHAGY ACTIVATION to Stay Young By This ONE Principle

Autophagy and hypoxia -

Although autophagy is known to be regulated mainly at the post-transcriptional level, transcriptional regulation has an important role in the induction of the process Moussay et al, We therefore investigated transcriptional regulation of autophagy-associated genes.

Hypoxia activates autophagy in GBM cells. D Western blot analysis showing p62 degradation upon hypoxia mean normalised to total protein content±s. Representative images were cropped from the same blots.

E Representative images show an increase in autophagosome formation upon hypoxia. Activation of autophagy was further visualised via increased conversion of LC3-I to LC3-II isoform under hypoxia Figure 2C.

To appropriately detect changes in the autophagic flux, experiments were performed in the absence and in the presence of the lysosomotropic agent chloroquine, which inhibits both the fusion of autophagosome with lysosome and lysosomal protein degradation. Interestingly, NCHk and U cells displayed high levels of LC3-II already in normoxia, suggesting their strong dependence on autophagy in normal conditions Figure 2C.

This is in accordance with the high sensitivity of U to chloroquine in both conditions Figure1G. Induction of autophagy by hypoxia was further confirmed by a decrease in p62 Figure 2D and an increase in the number of autophagosomes visualised via transient LC3-GFP transfection Figure 2E.

In conclusion, these data indicate that autophagy is induced under severe hypoxia in GBM cells. The heterogeneous sensitivity to autophagy inhibition corroborates with the differential basal level of autophagy in normoxia and further activation of autophagy in hypoxic GBM cells.

To further explore the GBM-specific response to hypoxia we focused on 98 specific regulators of autophagy positive and negative regulators, Supplementary Table S3.

Although the number of deregulated genes and the extend of deregulation was variable, we found four commonly deregulated genes shared between short-term and long-term hypoxia ATG9A , BNIP3 , BNIP3L and PIK3C3 ; Figure 3A ; Supplementary Table S3 , showing increased levels upon hypoxia.

BNIP3 and BNIP3L were previously associated with the autophagic response in hypoxic conditions Mazure and Pouyssegur, , whereas PIK3C3 is a well-known partner in the autophagy onset mechanism Munson and Ganley, Of note, MTOR , a negative regulator of autophagy and of PIK3C3, was significantly downregulated in 3 out of 4 GBM cultures Supplementary Table S3.

Interestingly, within the ATG family, only ATG9A was upregulated in all GBM cells Figure 3B ; Supplementary Table S3 , ATG2A was high only in 5 out of 8 conditions Supplementary Table S3.

The upregulation of ATG9A was confirmed by qPCR in GBM stem-like cells NCH, NCHk, NCHh, NCH, NCH and adherent cultures U87, U Figure 3C. ATG9A is specifically activated upon autophagic response to hypoxia. Venn diagrams reveal commonly deregulated genes. See Supplementary Table S3 for more autophagy-related genes.

C QPCR confirmed increased ATG9A expression in hypoxia. EZRIN was used as a reference mean±s. Interestingly, analysis of the ATG9A gene promoter revealed the presence of five hypoxia response elements HREs in close proximity to the canonical transcription start site, confirmed to be functional according to the TRANSFAC database Matys et al, ; Mole et al, Supplementary Table S4.

This was true also for BNIP3 , BNIP3L and PIK3C3 promoters, and is in line with the HIF-dependent regulation reported for the BNIP3 and BNIP3L Kothari et al, ; Mole et al, ; Slemc and Kunej, In summary, we show for the first time that ATG9A expression is strongly induced in hypoxic conditions, implicating ATG9A as a new player of hypoxia-dependent autophagic response in GBM.

Contrary to the control, ATG9A -depleted U87 cells did not increase the number of LC3-positive vesicles upon hypoxia Figure 4C , suggesting inefficient activation of autophagy. To examine the effect of ATG9A silencing on tumour growth in vivo , we implanted shATG9A NCHk and NCH cells into the brain of immunodeficient mice.

Of note, two of the autopagy-associated genes, ATG9A and BNIP3L , were included in our previously reported targeted shRNA screen, where we examined the essentiality of 55 genes for survival of GBM cells in vitro and in vivo Sanzey et al, Interestingly, ATG9A but not BNIP3L was also depleted both in vitro and in vivo Figure 4E , indicating that ATG9A is essential for general GBM cell survival.

Taken together, our data show that ATG9A is important for GBM growth both in normoxic and hypoxic conditions, and regulates activation of autophagy upon hypoxia.

Interfering with ATG9A expression efficiently blocks tumour growth in vivo. ATG9A knockdown decreases GBM cell proliferation and increases mouse survival. A QPCR confirmation of shATG9A knockdown mean±s. B Proliferation of shATG9A cells was decreased significantly in normoxic and hypoxic conditions mean±s.

C Representative images show lack of increased autophagosome formation upon hypoxia in shATG9 U87 cells. E Targeted in vivo shRNA screen in NCHk cells. Relative representation of respective shRNAs after selection pressure is presented as ratios compared with the original shRNA pool before selection baseline.

For detailed experimental setup, see Sanzey et al, Hypoxia is a characteristic feature of malignant gliomas and drives tumour progression by adaptive cellular responses including angiogenesis, changes in tumour metabolism, motility and survival Bertout et al, Increased hypoxia is also one of the escape mechanisms driving resistance to anti-angiogenic treatment in GBM.

Here we find that the autophagy pathway is strongly induced in GBM under hypoxia, and we identify ATG9A as a novel regulator of autophagy induction.

Inhibiting autophagy was shown to potentiate various anti-cancer therapies in vitro , including gliomas Kanzawa et al, ; Shingu et al, , where cells were subjected to external stress. Although, there are currently over 20 clinical studies involving the use of chloroquine and hydroxychloroquine in cancer treatment, in GBM both agents showed limited effect in non-toxic doses Sotelo et al, ; Rosenfeld et al, Here we show a significant increase in survival of GBM PDXs when chloroquine was administered as a single agent, although with different effective dose.

This is in accordance with the recent clinical trial showing dose-dependent inhibition of autophagy by hydroxychloroquine in GBM patients Rosenfeld et al, and suggests that dosing needs to be adapted to the specific patient tumour.

Of note, we show that certain tumour cells were sensitive to autophagy inhibitors also at normal oxygen, indicating a strong dependence on autophagy without additional environmental stress in a subgroup of GBM.

This heterogeneous response suggests that the genetic background, for example, PTEN deletion, p53 mutation or EGFR amplification, may differentially affect the extent of basal level of autophagy and of treatment response in GBM and that appropriate biomarkers may be required to efficiently stratify patients.

EGFR is known to negatively regulate autophagy Chen et al, through multiple signalling pathways, thus EGFR overexpression may partly explain the lower sensitivity of T16 tumours to anti-autophagic treatment.

Importantly, we find that bevacizumab treatment sensitised GBM cells to autophagy inhibition allowing to reach survival benefit at lower dose. This was confirmed in vitro , where hypoxia increased sensitivity of GBM cells to autophagy inhibitors.

The synergistic effect of bevacizumab was visible only when the anti-autophagy effect alone was mild or moderate at normal oxygen levels, but was masked if the autophagy inhibitor alone showed a strong effect. Interestingly, we have previously shown that bevacizumab leads to a lower number of mitochondria in tumour cells Keunen et al, , suggesting that mitophagy might be involved in the survival under hypoxia.

A previous study has shown efficacy of chloroquine in combination with bevacizumab in subcutaneous U87 tumours, but failed to observe a tumour suppressive effect with chloroquine used as a single agent Hu et al, This discrepancy may be due to the different tumour localisation and the heterogeneity in the GBM response to chloroquine described here.

Although non-specific effects of chloroquine cannot be excluded Maycotte et al, ; Maes et al, , we did not observe vessel normalisation upon chloroquine treatment.

In line with a previous study Chen et al, , normal astrocytes remained unaffected at the lowest chloroquine concentration affecting GBM cells, confirming more substantial dependence of tumour cells on autophagy.

We found that activation of autophagy in hypoxia was linked to transcriptional changes of numerous genes associated with autophagy, among which BNIP3 , BNIP3L , ATG9A and PIK3C3 were upregulated in all GBM cells.

BNIP3 and BNIP3L, while activated by HIF1 α , mediate autophagy by releasing Beclin1 from complexes with Bcl-2 and Bcl-X L Zhang et al, ; Bellot et al, Interestingly, within the ATG family, only ATG9A was transcriptionally activated in all GBM cells.

Contrary to other ATG family members such as ATG5 and ATG7 , but similarly to BNIP3 and BNIP3L , we identify ATG9A as potentially HIF1 α responsive gene. These transcriptional changes were observed also in GBM cells that exhibit high basal autophagy at normal oxygen levels, suggesting that specific upstream molecules such as FOXO3A are involved in the regulation autophagy pathway at different oxygen levels.

Pro-autophagic genes, such as Beclin1, ATG5, ATG7, BNIP3 and BNIP3L were previously found to be essential for autophagy in cancer cells Zhang et al, ; Mazure and Pouyssegur, Here we show that ATG9A also represents an important pro-survival molecule, with ATG9A depletion leading to a strong reduction of tumour growth, thus confirming the relevance of autophagy as a promising target for GBM treatment.

Of note, ATG7 knockdown displayed a therapeutic outcome only during anti-angiogenic treatment Hu et al, ATG9A was shown to be essential for autophagosome biogenesis and membrane maturation; however, its mode of action remains enigmatic.

Recent data suggest that the Pho—Rpd3 complex regulates expression of ATG9A and other ATG genes upon induction of autophagy Jin and Klionsky, and that ATG9A-containing vesicles are generated de novo upon starvation Yamamoto et al, Here we show that upon ATG9A depletion, GBM cells were not able to activate autophagy upon hypoxia.

We propose that the lack of autophagic activation upon hypoxia may be due to inhibition of de novo autophagosome synthesis. This is in accordance with a recent report, where ATG9A was shown to have a key role in autophagosome formation during hypoxic stress Weerasekara et al, Thus, ATG9A may become essential upon autophagy induction and an increased demand for new autophagosome membranes Orsi et al, In conclusion, our data support the notion that inhibiting autophagy represents an effective therapy in primary GBM, although it may be concentration and patient dependent.

Anti-autophagy treatment using genetic and pharmacological intervention was effective as a single treatment. However, currently available drugs, including chloroquine and hydroxychloroquine are non-curative in non-toxic doses and novel more potent agents will be necessary for GBM patients.

Drugs directly targeting essential proteins such as ATG9A may be of particular interest and a combination with anti-angiogenic therapy may be beneficial. Finally, the hypoxic microenvironment also contributes to immunoresistance and hypoxia-induced autophagy impairs cytotoxic T-lymphocyte-mediated cell lysis of tumour cells Noman et al, , and NK-mediated target cell apoptosis Baginska et al, ; Viry et al, Therefore, targeting autophagy in tumour cells may not only lead to increased tumour cell death but also sensitise tumours to immunotherapies.

This paper was modified 12 months after initial publication to switch to Creative Commons licence terms, as noted at publication.

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Download references. We thank Morgane Sanzey, Virginie Baus, and Amandine Bernard for technical assistance. We thank Dr Christel Herold-Mende Department of Neurosurgery, University of Heidelberg, Germany and Dr Uros Rajcevic National Institute of Biology, Ljubljana, Slovenia for providing cell lines.

This work was supported by the Luxembourg Institute of Health LIH , the Fonds National de la Recherche FNR of Luxembourg ESCAPE BM, AFR grand to SAAR and AD and the Fondation Cancer Luxembourg INVGBM.

Department of Oncology, NorLux Neuro-Oncology Laboratory, Luxembourg Institute of Health, Luxembourg City, L, Luxembourg. Faculty of Science, Technology and Communication, University of Luxembourg, Esch-sur-Alzette, L, Luxembourg.

Department of Oncology, Proteome and Genome Research Unit, Luxembourg Institute of Health, Luxembourg City, L, Luxembourg. Department of Oncology, Laboratory of Experimental Cancer Research, Luxembourg Institute of Health, Luxembourg City, L, Luxembourg. Department of Biomedicine, KG Jebsen Brain Tumour Research Center, University of Bergen, Bergen, N, Norway.

You can also search for this author in PubMed Google Scholar. Correspondence to Simone P Niclou. This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 4.

Supplementary Information accompanies this paper on British Journal of Cancer website. From twelve months after its original publication, this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 4. Reprints and permissions. Abdul Rahim, S. et al.

Regulation of hypoxia-induced autophagy in glioblastoma involves ATG9A. Br J Cancer , — Download citation. Revised : 07 June Accepted : 13 July Published : 10 August Issue Date : 05 September Anyone you share the following link with will be able to read this content:.

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Autophagy and hypoxia you for visiting nature. You are using Auhophagy browser version with hypoxiz support for Body cleanse recipe. To obtain the hy;oxia experience, we recommend you use Insulin sensitivity enhancement formula more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Hypoxia is a physiological stress that frequently occurs in solid tissues. However, the mechanisms underlying autophagy initiation upon hypoxia remains unclear.

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