Autophagy is required for GBM development

To test the effect of autophagy inhibition on KRAS-mediated gliomagenesis, we performed intracranial injection of DF-1 cells expressing RCAS viruses into neonate mice. Injection of DF-1 expressing KRAS:shLacZ RCAS viruses resulted in tumor formation in approximately 60% of the mice within 6 wk after injection (Fig. 2A–B) as reported previously.¹³ In contrast, the expression of shRNA targeting autophagy genes resulted in a dramatic reduction in KRAS-mediated tumor development (KRAS:shAtg7, ~10% of the injected mice developed tumors) or complete inhibition of tumor formation (KRAS:shAtg13 and KRAS:shUlk1). Overall, these results indicate that autophagy is required for gliomagenesis induced by KRAS expression.

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Figure 2.

Autophagy is required for GBM development. Mice were injected with RCAS vectors expressing KRAS in the presence of shRNA against LacZ, Atg7, Atg13 or Ulk1. (A) Kaplan Meier curve measuring tumor-free animals monitored up to 10 wk after injection with the indicated RCAS expressing cells. ***, p < 0 .001 (Log-rank Mantel-Cox test). (B) Animal numbers of those plotted in (A). (C) Representative H&E images of mouse brain sections showing GBM development in the indicated injections. (D) Tissue sections of normal or GBM brains were stained with antibodies to NES/nestin, or MKI67/Ki67 or with DAPI. (E) Immunocytochemical staining of brain sections using an antibody against ATG7. (F) Similar staining procedures using atg7 knockout (KO) or wild-type (WT) MEF cells to confirmed antibody specificity.

Having observed that some tumors did form in KRAS:shAtg7-injected mice (2 out of 21 mice), we assessed whether these tumors differed from the control (KRAS:shLacZ)-injected mice. We analyzed tissue sections by hematoxylin and eosin (H&E) staining and observed no difference in the tumor morphology between KRAS:shLacZ and KRAS:shAtg7 tumors (Fig. 2C). Similarly, there was no difference in NES/nestin (marker of GBM) or MKI67/Ki67 (proliferation marker) staining (Fig. 2D). Next we examined whether KRAS:shAtg7-derived tumors exhibited efficient reduction of ATG7 protein levels. Interestingly, immunohistological staining showed comparable ATG7 levels between shAtg7- and shLacZ-expressing tumors, suggesting that the tumor cells bypassed the Atg7 shRNA effect (Fig. 2E). We further confirmed the specificity of the anti-ATG7 antibody by staining mouse embryonic fibroblasts (MEFs) derived from wild-type or atg7 knockout mice (Fig. 2F). Overall, these observations further support the conclusion that the expression of autophagy-essential genes is required for GBM formation in the KRAS-RCAS/TVA model.

Autophagy is required for KRAS-driven oncogenic growth of glial cells

The lack of tumor formation upon RNAi-mediated inhibition of Atg7, Atg13 or Ulk1 suggests that autophagy is required for KRAS-driven gliomagenesis in vivo. However, these results do not distinguish whether the absence of autophagy impedes KRAS-induced transformation or growth of transformed cells. In order to understand the role of autophagy in gliomagenesis, we infected XFM glial cells (cdkn2a/ink4a-arf^−/-) with RCAS viruses co-expressing KRAS and shRNA against LacZ, Atg7, Atg13 or Ulk1 (as in Fig. 1B). We observed no growth defects upon autophagy inhibition in cells grown under normal culture conditions in monolayer. Interestingly, when cells were seeded in anchorage-independent conditions in soft agar, colony formation was strongly reduced in KRAS cells co-expressing shRNA targeting Atg7, Atg13 or Ulk1 compared to KRAS:shLacZ expressing cells (Fig. 3A). Therefore, autophagy appears to be critical for KRAS-driven glial transformation. Furthermore, we tested whether autophagy is required for clonogenic growth under suboptimal conditions by culturing cells in low serum (0.1% fetal bovine serum [FBS]), a condition mimicking poor angiogenic state in tumors. Under this condition, autophagy inhibition by Atg7-shRNA resulted in failure of clonogenic cell growth when cells were allowed to recover in full-growth medium containing 10% FBS compared to control cells (Fig. 3B–C). Overall, these studies indicate that autophagy is required for both oncogenic transformation of KRAS-expressing glial cells and their sustained viability under stressful conditions associated with gliomagenesis.

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Figure 3.

Inhibition of autophagy suppresses oncogenic growth and growth signaling pathways in KRAS-expressing glial cells. Primary glial cells (XFM) were transduced with RCAS viruses co-expressing KRAS along with the indicated shRNA and grown under the indicated conditions. (A-B) Transduced XFM cells were grown in (A) soft agar or (B) low serum (0.1% FBS) for 2 wk followed by incubation in full growth medium for 1 week. (C) Representative images of cell morphology for cells treated as in (B). (D) No induction of apoptosis was observed (measured by cleaved CASP3) in cells cultured in 0.1% FBS for the indicated time points. Staurosporine (STS) treatment in KRAS:shLacZ cells was used as a positive control for CASP3 activation. (E) Cells grown in low serum (0.1% FBS) for the indicated times and cell lysates were analyzed by western blot using antibodies against the indicated proteins. (F) KRAS:shLacZ- or KRAS:shAtg7-expressing XFM cells were grown under hypoxic conditions (0.5% O₂) for 72 h and cell lysates analyzed. Statistical analyses are shown of 3 independent experiments performed in triplicates including error bar (SEM values). *, p < 0 .05; **, p < 0 .01, ***, p < 0 .001 (Student t test, unpaired 2-tailed).

We further tested the underlying mechanism for the lack of clonogenic growth in autophagy-inhibited cells by analyzing growth signaling and survival pathways. We observed neither morphological changes indicative of cell death induction in cells cultured under low-serum condition (0.1% FBS) nor CASP3/caspase-3 activation, indicating that the difference in growth capacity was not due to apoptotic cell death (Fig. 3D). Next, we tested whether growth signaling pathways were impeded by Atg7 knockdown. When grown in normal culture medium, the phosphorylation status of signaling factors AKT, MAPK1/ERK2-MAPK3/ERK1, and RPS6 was comparable in KRAS:shLacZ- and KRAS:shAtg7-expressing cells, indicating that autophagy was dispensable under this condition. Interestingly, under low-serum conditions, while KRAS:shLacZ-expressing cells could maintain modest levels of AKT, MAPK1/3 and RPS6 phosphorylation, KRAS:shAtg7-expressing cells were more susceptible to serum withdrawal (Fig. 3E). We further confirmed the role of autophagy in maintaining growth signaling by culturing cells in hypoxic conditions (a critical aspect of GBM). Autophagy was significantly induced in KRAS:shLacZ cells grown under low oxygen conditions (0.5% O₂, Fig. 3F). Importantly, both AKT and MAPK1/3 phosphorylation were significantly lower in KRAS:shAtg7 cells compared to control cells, indicating that growth signaling under hypoxia was compromised in the absence of autophagy. In contrast, no significant effects were observed on RPS6 phosphorylation, suggesting that the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) complex 1 (MTORC1) signaling pathway may be dispensable under these conditions. Overall, the above results strongly suggest that autophagy is required to maintain growth signaling pathways in cells deprived of growth factors or grown under hypoxia.

Western blot and antibodies

For western blot analyses, cell lysates were prepared in RIPA buffer (10 mM Tris pH 7.4, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 0.1% sodium dodecyl sulfate, 1% Triton-X100 [Sigma, T9284], 1 mM 2-mercaptoethanol, 0.5% sodium deoxycholate and 10% glycerol) and analyzed by SDS-PAGE as described previously.^7,29 The following antibodies were used: anti-LC3 (Sigma, L7543); anti-ACTB/β-actin (Sigma, A5316); anti-ULK1 (Sigma, A7481); anti-ATG7 (Santa Cruz Biotechnology, clone H300, sc-33211; or Sigma, A2856); anti-pan-RAS (EMD Millipore, OP40); anti-RAS G12D mutant specific (Cell Signaling Technology, 14429); anti-ATG13 (Sigma, SAB4200100); anti-TUBG/γ-tubulin (Sigma, GTU-88); anti-HIF1A/HIF1α (R&D Systems, MAB1536); anti-phosphorylated (p)-AKT (Ser473; Cell Signaling Technology, 4060); anti-total AKT (Cell Signaling Technology, 9272); anti-p-MAPK1/ERK2-MAPK3/ERK1 (Thr202/Tyr204; Cell Signaling Technology, 4370); anti-total MAPK1/ERK2-MAPK3/ERK1 (Cell Signaling Technology, 9102); anti-pRPS6/S6 (Ser235/236; Cell Signaling Technology, 4858); anti-total RPS6/S6 (Cell Signaling Technology, 2217); anti-CASP3/caspase-3 (Cell Signaling Technology, 9665); anti-RB1/p105 (BD Biosciences, 554136); anti-TRP53/p53 (Cell Signaling Technology, 2524); anti-CDKN1B/p27 (Cell Signaling Technology, 2552); anti-IL6/interleukin 6 (R&D Systems, BAF-406); anti-IL1B/interleukin 1 β (R&D Systems, AF-301-NA).

Injections

All animal experiments were done in accordance with protocols approved by the Institutional Animal Care and Use Committees of Memorial Sloan Kettering Cancer Center and followed NIH guidelines for animal welfare. The RCAS/TVA system used in this work to induce gliomas in vivo in immunocompetent mice has been described previously.³⁰ N/TVA;cdkn2a/ink4a-arf)^−/-;Pten^fl/fl mice were used for the RCAS mediated gliomagenesis in this study.¹³ Briefly, DF-1 cells were transfected with the relevant RCAS viral plasmid using Lipofectamine2000 (Invitrogen, 11668019) according to the manufacturer's protocol. The cells were regularly maintained for at least 3 passages for propagation of the RCAS viruses to entire cells. Cells were then used for injection into murine brain. Newborn pups were injected intracranially with 1 μL of ~1×10⁵ DF-1 cells. Then mice were monitored until they developed symptoms of GBM such as lethargy, poor grooming, weight loss, dehydration or macrocephaly. GBM incidence or absence was confirmed by H&E staining of brain sections in all injected animals. Kaplan-Meier analysis demonstrating symptom-free survival in murine gliomas was performed using the Prism software (GraphPad).

Vector constructs

For the generation of the RCAS-shRNA vector, shRNAs were initially assembled in the pSUPER.retro vector (OligoEngine, VEC-PRT-0002). The shRNAs containing the H1 promoter were PCR-amplified and then inserted into the RCAS-Y vector (generated and provided by Dr Yi Li, Baylor College of Medicine, Texas)³¹ using NotI and PacI restriction sites. KRAS was subsequently cloned using NotI restriction sites. The shRNA target sequences are as follows: Atg7 5′ CACATAGCATCATCTTTGA; Atg13 5′ GAGAAGAATGTCCGAGAAT; Ulk1 5′GAGCAAGAGCACACGGAAA.

Immunocytochemistry

Mouse brains harvested from animals were fixed in 10% formalin for 24–72 h and then transferred to 70% ethanol. Samples were then paraffin-embedded, sectioned and stained with H&E by Histoserv Inc., Maryland. Similarly, MEFs derived from wild-type or atg7 knockout cells were pelleted and processed for paraffin embedding. For immunocytochemistry staining, paraffin-embedded sections were treated with xylene twice for 10 min each before being sequentially hydrated in decreasing concentrations of ethanol. Epitope retrieval was performed by incubating slides in 10 mM sodium citrate buffer, pH 6 for 15 min at 95°C then allowed to cool for 20–45 min. Subsequently, slides were blocked with TBST (150 mM NaCl and 10 mM Tris pH 7.5, 0.1% Tween 20 [Sigma, P5927] + 5% BSA [Thermo Fisher Scientific, BP1605-100]) for 45 min, and incubated with primary antibodies overnight at 4°C. Subsequently, slides were incubated with Alexa fluorescent secondary antibodies (Life Technologies, A-11012 and A-11032) and images acquired using a Nikon Eclipse Ti-U Confocal Microscope. The antibodies used in this study are anti-ATG7 (Sigma, A2856), anti-MKI67/Ki67 (Vector Labs, VP-K451), and anti-NES/nestin (BD Biosciences, 556309).

Colony formation assay

XFM cells were seeded in 6-cm dishes; 24 h later cells were cultured in 0.1% FBS for 2 wk (with medium replenished after 1 wk) followed by growth in 10% FBS for a further 1 wk. Cells were then fixed with 10% formaldehyde and stained with Giemsa (Sigma, GS500) to visualize colonies.

Hypoxia

For hypoxia experiments, cells were incubated in a Whitley H35 Hypoxystation set to 37°C and 0.5% O₂. 72 h later, cells were lysed inside the Hypoxystation and analyzed by western blotting.

Soft agar assay³²

XFM cells were seeded at 10,000 cells/well in a 6-well dish. Cell suspension (final volume 1.5 mL) containing 0.4% low gelling agarose (Sigma, A4018) in full growth DMEM was overlaid with a layer of 0.5% agarose and left to solidify at 4°C. Cells were then incubated at 37°C, fed weekly with 0.4% agarose-containing DMEM and analyzed after ~3 wk by staining with 0.02% Iodonitrotetrazolium chloride (Sigma, I10406). Colonies were quantified using an Optronix Gelcount (Oxford Optronix). All assays were conducted in triplicates in 3 independent experiments.

Senescence-associated β-galactosidase assay (SA-GLB1)²³

Cellular senescence was measured using an SA-GLB1 assay. Briefly, cells were fixed with 0.5% glutaraldehyde then incubated with X-gal staining solution, pH 6.0 (1mM MgCl₂ phosphate-buffered saline [Fisher BioReagents, BP399], X-gal [Thermo Fisher Scientific, R0941], 0.12 mM K₃Fe[CN]₆ [Sigma, 60299], 0.12 mM K₄Fe[CN]₆ [Sigma, 60279]) overnight at 37°C. Images of cells were taken using a Nikon Digital Sight DS-L3.

BrdU incorporation assay

For the BrdU incorporation assay, cells were pulsed with 50 μM BrdU (Sigma, B5002) for 18 h followed by fixation with 3.7% paraformaldehyde and permeabilization with 0.2% Triton-X100. Cells were blocked with 0.2% gelatin-fish (Sigma, G7765) in 5% BSA-phosphate-buffered saline and incubated with anti-BrdU primary antibody (1:2000; BD Biosciences, 555627), 0.5 U/L DNase (Sigma, D4527), and 1 mM MgCl₂ in blocking solution. Subsequently, slides were incubated with Alexa secondary antibodies (Life Technologies, A-11001) and 1 μg/ml DAPI; images were acquired using ImageXpress and analyzed using MetaXpress software.

Metabolite measurement

XFM cells were grown in 0.1% FBS for 2 wk and fresh 0.1% FBS medium was added and cells grown for a further 3 or 5 d. Alternatively, cells grown in 10% FBS were analyzed after 3 d of culture. Metabolites were extracted from the medium using ice-cold extraction buffer (50% methanol, 30% acetonitrile). Extracted metabolites were separated on a Zic-pHILIC column (Merck Millipore) using a Thermo Ultimate BioRS HPLC with a single step linear gradient of 1095%– A over 20 min (mobile phases were [A] 20 mM ammonium carbonate [B] acetonitrile). Metabolites were eluted into a Q-Exactive mass spectrometer (Thermo Fisher Scientific) using a flow rate of 300 µl/min. Metabolite masses were acquired in negative mode within the range of 78–250 m/z. Relative metabolite abundance was then determined by integrating the ion peak area (Quan Thermo Xcalibur) and normalized to cell number.