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Autophagy is associated with chemoresistance in neuroblastoma

Autophagy is associated with chemoresistance in neuroblastoma Background: Neuroblastoma (NB) is a frequent pediatric tumor characterized by a poor prognosis where a majority of tumors progress despite intensive multimodality treatments. Autophagy, a self-degradative process in cells, could be induced by chemotherapy and be associated with chemoresistance. The aim of this study was to determine whether: 1) autophagy is present in NB, 2) chemotherapy modified its levels, and 3) its inhibition decreased chemoresistance. Methods: Immunohistochemical stainings were performed on samples from 184 NB patients in order to verify the expression of LC3B, a specific marker for autophagy, and Beclin 1, a positive regulator of autophagy. In addition, we performed an in vitro study with six NB cell lines and six drugs (vincristine, doxorubicin, cisplatin temozolomide, LY294002 and syrolimus). Inhibition of autophagy was performed using ATG5 knockdown cells or hydroxychloroquine (HCQ). Cell survival was measured using the MTT cell proliferation assay. Autophagy was detected by monodansylcadaverine, confocal microscopy and Western blot. In vivo study with tumor xenografts in NSG mice was performed. Results: Our results have indicated that autophagy was present at low levels in NB and was not a prognostic factor, while Beclin 1 was highly expressed in children with poor NB prognosis. However, autophagy levels increased after chemotherapy in vitro and in vivo. Tumor progression was significantly decreased in mice treated with a combination of HCQ and vincristine. Conclusions: Taken together, autophagy is present in NB, induced by chemotherapy and associated with chemoresistance, which is significantly reduced by its inhibition. Therefore, targeting autophagy represents a very attractive approach to develop new therapeutic strategies in NB. Keywords: Neuroblastoma, Autophagy, Chemoresistance, Hydroxychloroquine Background resistance to high-dose chemotherapy indicate that NB is Neuroblastoma (NB) is the most common and deadly ex- specifically associated with chemoresistance [7]. tracranial solid tumor in children [1, 2]. Survival of children Autophagy is a ubiquitous self-degradation process that older than one year of age with advanced NB is poor (only involves the degradation and recycling of cellular cytoplas- 34%), despite aggressive treatments [3, 4]. High-dose mic constituents through the lysosomal pathway. Damaged chemotherapy with autologous hematopoietic stem cell or misfolded proteins or organelles are first sequestered in transplantation significantlyimprovesthe prognosisof double-membrane vesicles, known as autophagosomes, metastatic NB [5, 6], but this treatment carries with it a before fusing with lysosomes, where their contents are de- high risk of adverse effects [4]. Poor global survival and graded by lysosomal proteases [8–10]. Autophagy is a com- plex and multistep process involving the autophagy-related proteins (ATG) [8]. ATG5 is a protein involved in the early * Correspondence: hsartelet@chu-grenoble.fr Research centre of the Sainte Justine university hospital, Montreal, QC, stages of autophagosome formation and plays an essential Canada role in the maturation of autophagosomes [11], with assist- Department of pathology and cellular biology, Université de Montréal, ance from LC3 [8]. Low levels of autophagic activity are Montreal, QC, Canada Full list of author information is available at the end of the article © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Belounis et al. BMC Cancer (2016) 16:891 Page 2 of 14 commonly observed under normal conditions, presumably System (INSS) [24]. Treatment was assigned according preserving normal cellular homeostasis [12, 13]. Prolonged to the risk group on the basis of the patient’s age at time autophagy may result in type 2 (autophagic) programmed of diagnosis, the INSS stage, the histoprognosis, the cell death [12, 14]. The activation of autophagy is measured ploidy and MYCN amplification status (v-myc avian by the ratio between LC3II on the autophagosome mem- myelocytomatosis viral oncogene neuroblastoma derived brane and LC3I in the cytoplasm which can be detected by homolog). With formalin-fixed and paraffin-embedded Western-blot [15] or by immunohistochemistry [16]. Beclin samples, a tissue microarray (TMA) was constructed 1 is also a marker and a positive regulator of autophagy. using four representative NB tumor tissue cylinders with The regulation of autophagy by the PI3K/AKT pathway a 0.6 mm diameter. TMA blocks contained not only 184 is very complex. Recently, an AKT inhibitor was reported primary tumors but also 47 paired metastases (42 lymph to induce autophagy with a radiosensitizing effect [17]. Au- nodes and 5 hepatic metastases). Among the 184 tophagy is regulated by both class I and III PI3K pathways tumors, 19 tumors were tested by Western blot, proteins [18, 19]. mTOR serves as a metabolic sensor that coordi- coming from the lysate of frozen samples. nates cross-talk between nutrient availability and autophagy [19]. On the other hand, class III PI3K in conjunction with Immunohistochemistry Beclin 1 positively regulates autophagy [18, 19]. Immunohistochemistry was performed on the sections of In cancer, autophagy plays a dual role by either activating the TMA blocks or of tumors developed in the mouse cell death and inhibiting tumor progression or promoting model. The Ultraview Universal DAB detection kit cell survival [20]. In the early stages of carcinogenesis, (Ventana, Ventana medical system, Tuscon, AR) was used. autophagy acts as a primary tumor suppressor and inhibits Antibodies against phospho-AKT (1/100, S473-r, Santa tumor progression [21]. However, autophagy can also Cruz biotechnology, CA), phospho-mTOR (1/100, 49 F9, confer tumor cells the ability to resist to ionizing radiation Cell Signaling, CA), LC3B (1/1000, ab51520 abcam, [22] as well as to chemotherapy [23]. Cambridge UK) or Beclin 1 (1/250, ab55878 abcam) were The observation of increased cell survival associated with applied for 30 min. DAB was used as a chromogen and higher autophagy activity following therapy has led to the hematoxylin as a counterstain. Normal mouse or rabbit development of strategies combining autophagy inhibitors IgG at the same concentration as the primary antibody to current anticancer treatments. In this context, chloro- were used as negative control and synaptophysin (1/100, quine (CQ) or its derivate hydroxychloroquine (HCQ), sen- Polyclonal, SP11, Thermofisher Scientific) as positive sitizes tumor cells to anticancer therapies. Indeed, CQ and control (Additional file 1: Figure S1). Two investigators HCQ block the processing and maturation of autophagy blinded for clinical data independently evaluated vacuoles (autophagolysosomes) by inhibiting lysosomal ac- immunostaining in samples containing more than 100 NB tivity [23]. Some data suggest that autophagy inhibition and cells. Immunostaining scores were established by a semi- autophagosome accumulation both contribute to the quantitative optical analysis assessing the percentage of accelerated cell death induced by HCQ [23]. positive cells in each sample: 0 = all cells negative, 1 + = 1 The aim of the present study is to demonstrate the to 25%, 2 + = 26 to 50%, 3 + = 51 to 75% and 4+ more than presence of autophagy in NB, its activation by chemo- 75% of positive tumoral cells. therapy and its correlation with chemoresistance. TUNEL Methods On the sections of TMA, a terminal deoxynucleotidyl Study design and patients transferase-mediated dUTP nick end-labeling (TUNEL) Study cases were selected upon the following inclusion assay (In situ cell death detection kit, POD (Roche)) was criteria; 1) a diagnosis of NB had been made between used to identify double-stranded DNA fragmentation, char- July 1988 and March 2008, 2) human subject research acteristic of DNA degradation due to apoptosis. Briefly, tis- (tissue samples) was approved by the Research Ethics sue slides were deparaffinized. The slides were then treated Board of the Sainte-Justine University Health Center. with 0.1% of Triton X-100 (Sigma, X-100). The slides were Written consent has been obtained from patient then incubated with terminal deoxynucleotidyl transferase guardians, and 3) adequate specimen material has been followed by peroxidase-conjugated anti-digoxigenin anti- collected for study purposes. 184 patients with NB were body. Finally, the slides were stained with DAB. Methyl included in our study. The patients were treated and green was performed as the counter-stain. Slides were followed up at Sainte-Justine University Health Center scanned using a customized, computer-controlled micro- (Montreal, Canada). Thirty-one out of 184 total NB scope (Axio Imager M1; Zeiss, Oberkochen, Germany). cases were identified from routine provincial (Quebec, The percentage of positive neuroblasts for TUNEL was also Canada) mass screening efforts. Tumors were classified calculated by dividing the number of stained nuclei by the according to the International Neuroblastoma Staging total numbers of neuroblasts and multiplying by 100. Belounis et al. BMC Cancer (2016) 16:891 Page 3 of 14 Table 1 Patients clinical data Variable LC3B Beclin-1 No (%) No % Mean p No % Mean p Intensity Intensity All patients 184 148 80 0.83 153 83 1.03 Age Median (Range), months 26 (0–151) <365 days 88 (48) 73 83 0.92 70 80 0.83 ≥365 days 96 (52) 74 77 0.75 ns 84 87.5 1.25 <0.01 Stage 1 53 (29) 47 88 0.6 ns 48 90 1.30 ns 2 35 (19) 25 71 0.64 27 77 0.8 3 23 (12) 21 91 0.94 20 86 1.14 4 58 (32) 52 89 1.12 47 81 0.97 4S 15 (8) 11 73 0.34 0.02 11 73 0.72 ns MYCN status Amplified 14 (10) 13 87 0.81 11 73 0.87 Non Amplified 127 (90) 103 81 0.50 ns 105 82 0.43 ns Unknown 43 Type of NB Standard 149 (81) 133 89 0.96 126 84 1.19 Mass screening 35 (19) 29 82 0.8 ns 27 77 0.46 <0.001 Type of samples Primary tumors 184 148 80 0.83 153 83 1.03 Metastases 47 41 87 0.78 ns 37 79 0.69 <0.01 Cell lines medium for 2 days before selection for stable expression of Six NB cell lines were studied: SK-N-DZ (ATCC, Manassas, theshRNA by growinginculture mediacontaining VA), SK-N-SH (ATCC), SK-N-FI (ATCC), IGR-N91 and puromycin (5 μg/mL)for at least2weeks. IGR-NB8 cells from Gustave Roussy Institute (Villejuif, France), and NB-10 (St. Jude Children’sHospital, Memphis, GFP-LC3 transfection and confocal microscopy TN). MYCN amplification is present in NB-10, SK-N-DZ The cell line IGR-N91 was transfected with GFP-LC3 and IGR-N91 cells. The cells were cultured in Dulbecco’s (Millipore’s LentiBrite TM GFP-LC3 lentiviral Biosensor) modified Eagle medium (DMEM), 10% fetal bovine serum for monitoring autophagosome formation. IGR-N91 cells (FBS) and 1% penicillin-streptomycin at 37 °C in a 5% CO were seeded at 4 × 10 /well into eight-well chamber slides atmosphere. (Thermo Scientific, Rochester, NY) to achieve 70% confluence. After 24 h, the cells were transfected with Knockdown of ATG5 expression by lentivirus-delivered lentivirus containing a version of GFP-LC3 at 37 °C, 5% shRNA CO for 24 h. At 48 h post-transfection, the medium was TRC Lentiviral Human ATG5 and eGFP shRNA vectors changed to DMEM, 1% fetal bovine serum (FBS) and 1% (ATG5: accession #NM_004849, eGFP: accession # penicillin-streptomycin, the cells were washed three times RHS4459) were purchased from Open Biosystems, with PBS and visualized with the Ultraview Vox Confocal Rockford, IL. Lentiviral vectors were produced using HEK Imaging System (Perkin Elmer). The autophagosome 293 T cells by PEG (polythylenimine linear, Polysciences volume in transfected cells was evaluated with Imaris 7.7.2 inc) transfection of ATG5 or eGFP shRNA plasmid software (Oxford Instruments Company). together with the third-generation packaging plasmids pMDL, pRev and pV-SVG (Open Biosystems). To generate Cell proliferation assay human ATG5-knockdown cells, IGR-N91 cells were Cell viability was determined by the MTT test. Cells transduced with lentivirus expressing shATG5 or sheGFP were plated in a 96-well culture plate at a density of for control. Transduced cells were cultured in fresh 5x10 /well over night. The cells were treated with Belounis et al. BMC Cancer (2016) 16:891 Page 4 of 14 Fig. 1 (See legend on next page.) Belounis et al. BMC Cancer (2016) 16:891 Page 5 of 14 (See figure on previous page.) Fig. 1 Evaluation of the level and the regulation of autophagy in NB and its correlation with apoptosis. A Autophagy was evaluated in TMA sections from NB tumor samples by immunohistochemistry using antibodies anti-LC3B (A.a,b) or anti-Beclin 1 (A.c). The expression levels of pmTOR (A.e) and pAKT (A.f), two autophagy-regulating proteins, were also studied on the same samples. Apoptosis was tested by TUNEL (A.d). Scale bars: 100 μm. B Immunoblotting analysis was performed on protein lysates from different frozen matched tumor samples with antibodies against either LC3B or Beclin 1 for autophagy and against cleaved caspase-3 for apoptosis. C The expression of LC3II and TUNEL positivity were semi-quantified according to the stage of tumors. D The ratio LC3II/LC3I and cleaved caspase-3 expression were evaluated by densitometry using tumor samples various conditions for 24 h then proliferation was mea- or with the association of the two drugs and then analyzed sured by the (3-[4,5dimethyl-2-thiazolyl]-2,5-diphenyl- with confocal microscopy for 7 h. 2H-tetrazolium bromide) MTT cell proliferation assay (Cell Titer 96 Non-Radioactive Cell proliferation Assay, Western Blot Promega) according to manufacturer’s instructions. Protein extracts were prepared from frozen tumor Absorbance was measured at 570 nm using the Spectra tissues of 19 patients and cultured cell lines. Cell lysis Max 190 microplate spectrophotometer (Molecular buffer with protease inhibitor cocktail (10 mM Tris–HCl Devices, Sunnyvale, CA). Assays were performed in pH 7.4, 150 nM NaCl, 1 mM EDTA, 1 mM EGTA, 1% triplicate. Relative cell viability (percent of control) was Triton X-100, 0.5% NP-40, 1 mM sodium orthovanadate, calculated using the equation: (mean OD of treated 1 mM sodium fluoride) was added to each sample. Equal cells/mean OD of control cells) × 100. For each time amounts of protein (20 μg) from cell lysates were solubi- point, the treated cells were compared with control cells lized in Laemmli sample buffer, boiled for 5 min, sepa- that had been treated with vehicle only. rated by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes, blocked 1 h at room Monodansylcadaverine (MDC) test temperature with TBS buffer (20 mM Tris–HCl, pH 7.4, To correlate cell survival with the presence of autopha- 150 mM NaCl) containing 3% bovine serum albumin, gic vacuoles, cells were incubated with the autofluores- and incubated with primary antibody overnight at 4 °C. cent agent monodansylcadaverine (MDC) at 0.05 mM in Immunoreactive bands were revealed following 1 h PBS (Sigma) for 10 min at 37 °C. MDC has been incubation with horseradish peroxidase-conjugated reported to specifically label autophagic vacuoles [25]. anti-rabbit or anti-mouse antibodies, and the signals MDC was then replaced with 200 μl of PBS and finally were visualized with an enhanced chemiluminescence replaced by 100 μl of Tris-Triton (Tris 10 mM pH 8.0, (ECL) detection system (PerkinElmer, Waltham, MA). 0.1% Triton X-100). Fluorescence was measured with The primary antibodies used for this study were: β-actin EnVision 2104 multilabel reader (Perkin Elmer) with an (13E5; diluted 1:5000, Cell Signaling, Danvers, MA), emission filter of 525 nm at 380 nm. mouse polyclonal anti-human PARP-1 (Ab-2; 1:600; Calbiochem, Billerica, MA), rabbit polyclonal anti- Cell treatments human Beclin 1 (1/1000, ab55878 abcam, Cambridge The MTT and MDC tests were performed on cell lines UK), rabbit polyclonal anti-human SQSTM1/p62, LC3B with or without transfection of ATG5 shRNA vector and and ATG5, rabbit monoclonal anti-human Cleaved treated with different concentrations of drugs for 24 h at Caspase-3 (Asp175), mTOR (7C10), phospho-mTOR 37 °C: temozolomide (Schering Plough inc, 0.1–1000 μM), (Ser2448, D9C2), Akt and phospho-Akt (Ser473), (all vincristine (Mayne Pharma USA inc, Paramus NJ, diluted 1:1000, Cell Signaling, Danvers, MA). A densi- 1–10000 nM), doxorubicin (Pfizer Inc Kirkland, Canada, tometry analysis with the Kodak ID 3.6 software was 0.005–50 μM) and cisplatin (Mayne Pharma USA inc, used to calculate the relative expression of Cleaved Paramus NJ; 0.015–150 μg/ml), LY294002, specific Caspase-3 and the ratio between LC3B-II and LC3B-I. inhibitor of AKT (Calbiochem Darmstadt, Germany, 0.05–500 μM) and rapamycin, specific inhibitor of mTOR Animal experiments (Pfizer Inc Kirkland, Canada 0.1–1000 nM). Finally, cells NOD/LtSz-scid/IL-2Rgamma null mice (NSG) were pur- were treated with varying concentrations of cisplatin chased from Jackson Laboratory (Bar Harbor, ME, USA). (0.15–75 μg/mL) and doxorubicin (0.005–5 μg/mL) in the Animal experiments were approved by the CEEA26 presence and absence of HCQ (30 μM) for 48 h. The com- Ethics Committee (approval number: 2013–099) and pound concentrations resulting in 50% inhibition of cell carried-out under the conditions established by the viability (IC ) were determined using GraphPad Prism 6 European Community (Directive 2010/63/UE). All mice software. The cell line IGR-N91 transfected with GFP-LC3 were housed in ventilated cages under standard condi- were treated with vincristine (1 μM), with HCQ (30 μM) tions of controlled temperature and humidity, and Belounis et al. BMC Cancer (2016) 16:891 Page 6 of 14 Fig. 2 (See legend on next page.) Belounis et al. BMC Cancer (2016) 16:891 Page 7 of 14 (See figure on previous page.) Fig. 2 Chemotherapy induces autophagy in vitro and in vivo. The effect of chemotherapeutic agents on the autophagy of NB cell lines and tissues was analyzed. a NB-10 cells were treated with increasing concentrations of temozolomide, rapamycin, cisplatin, vincristine, doxorubicin or LY294002 for 24 h followed by MTT and MDC assays to measure cell viability and autophagy levels, respectively. Results are expressed as percentage of the corresponding control and represent mean ± SEM of 3 independent experiments (***: P < 0.001). b Cell viability and MDC specific autophagy were measured after IC50 treatment of different NB cell lines with chemotherapeutic agents. Results are expressed as percentage of the corresponding control and represent mean ± SEM of 3 independent experiments (***: P < 0.001). c Immunobloting analyses were performed on protein lysates from SK-N-SH and IGR-N91 cells treated with 0.1 to 10 μM of vincristine. Anti PARP-1, anti-LC3B, anti-p62, anti-mTOR, anti-pmTOR antibodies were used. β-actin was used as a loading control. d Tumor xenografts developed from SK-N-DZ or IGR-N91 cells were treated with cisplatin or vincristine. Slides were immunostained with anti-LC3B antibody. Scale bars: 100 μm exposed to a 12-hourly light/dark cycle. They were Autophagy and neuroblastoma provided with standard diet and water ad libitum. First, The analysis of LC3 immunostaining clearly showed two NSG were established in 6 to 8 week-old mice by sub- types of positivity: uniform cytoplasmic staining, corre- cutaneous injection of 5 × 10 IGR-N91 in the left flank. sponding to LC3I (Fig. 1Aa) and a punctate cytoplasmic Mice were monitored twice a week for tumor growth. LC3-staining pattern specific to LC3II and therefore to Clinical monitoring of mice (weight, vital signs, behavior autophagy (Fig. 1Ab). Furthermore, autophagy was and abdominal palpation) were carried out once a week. frequent in NB (80% of the tumors) but with a very low When the palpable tumor reached 100 mm (measured intensity (mean intensity 0.83). These data were con- with calipers), 6 mice/group received a dose of vincris- firmed by Western blot analysis where LC3II expression tine (0.4 mg/kg) [26] or HCQ (60 mg/kg) or an associ- was found in 12 out of 19 tumors (63%) (Fig. 1B). The ation of vincristine and HCQ at the same concentration cytoplasmic expression of Beclin 1 was also expressed in was injected daily for 9 consecutive days. Six control a majority of NB (83%) but with a low intensity (mean mice received (100 μL) of saline solution per day. Mice intensity 1.03) (Fig. 1Ac, Table 1). These data were were euthanized at the onset of clinical signs or when confirmed by Western blot analysis showing Beclin 1 bearing tumors of 1000 mm or at the end of the expression in 17 out of 19 tumors (90%) (Fig. 1B). treatment. The LC3II expression was significantly higher in Stage Secondly, five millions of SK-N-DZ cells (100 μL) were 4 (mean intensity 1.12) than in Stage 4S (mean intensity injected s.c. in the left flank. Mice were treated with 0.34) (Table 1, p = 0.02), confirmed by Western blot, vincristine (0.4 mg/kg) or cisplatin (Mayne Pharma, where LC3II was undetectable in specimen from Stage Canada) i.p. at 8 mg/kg for 4 days [27, 28]. Mice 4S patients. Interestingly, the level of apoptosis detected receiving PBS at the same time were used as a negative by TUNEL (Fig. 1Ad) and by cleavage of caspase-3 was control. In all in vivo experiments, mice were euthanized inversely correlated with autophagy, particularly in 10 days after the end of treatment or when tumor size Stages 4 and 4S (Fig. 1C and D). However, expression reached 1000 mm . Tumors were collected and levels of Beclin 1 were significantly higher in patients immunohistochemistry with LC3B antibody was older than one year at diagnosis compared to younger performed. patients (p < 0.001) (Table 1), in primary tumors than in metastasis (p < 0.01) and in standard NBs than in tumors coming from mass screening (p < 0.001), demonstrating Statistical analysis that Beclin 1 is highly expressed in NB with poor Association tests were performed with the use of Fisher’s prognosis. Among the major proteins present in the exact test. Spearman correlation values (rho) were used AKT pathway, there was a significant negative correl- to compare the expression between LC3-II and pmTOR, ation between LC3II and pmTOR (Fig. 1Ae) (rho = pAKT and between Beclin 1 and pAKT. Statistical ana- −0.22, p < 0.01) as well as pAKT (rho = −0.22, p < 0.01) lyses were performed using GraphPad Prism 6 software. and between Beclin 1 and pAKT (Fig. 1Af) (rho = −0.13, P values of less than 0.05 were considered to indicate p < 0.01), suggesting that autophagy is activated through statistical significance. AKT pathway inhibition. Results Chemotherapy induces autophagy in neuroblastoma cells Characteristics of patients The formation of autophagosomes revealed by MDC Clinicopathological characteristics of the 184 patients indicates a significant increase of autophagy following and tumors are detailed in (Table 1). These patients had treatment with increasing concentrations of cisplatin, a median follow-up period of 67 months (range vincristine and doxorubicin (Fig. 2a). Temozolomide newborn-174 months) and a median age at the time of didn’t induce high levels of autophagy even at high con- diagnosis to be 26 months (range newborn-151 months). centrations. AKT pathway inhibitors, such as LY294002 Belounis et al. BMC Cancer (2016) 16:891 Page 8 of 14 Fig. 3 (See legend on next page.) Belounis et al. BMC Cancer (2016) 16:891 Page 9 of 14 (See figure on previous page.) Fig. 3 Inhibition of autophagy sensitizes NB cells to chemotherapy. IGR-N91 cells were transduced with ATG5-shRNA lentivirus to knockdown ATG5 gene expression and thereby inhibit autophagy or with eGFP-shRNA, used as a control. a Protein immunoblotting using anti-ATG5 and anti-LC3B antibodies was performed to demonstrate ATG5 gene knockdown and LC3 expression on IGR-N91-transduced cells or not (WT). β-actin was used as a loading control. kd b A MDC test for autophagy was performed on ATG5 and control cells after treatment with increasing concentrations of vincristine. c Cell viability of kd ATG5 and control cells was measured with a MTT assay following treatment with increasing concentrations of vincristine, doxorubicin or cisplatin. Results are expressed as percentage of corresponding control and represent mean ± SEM of 4 independent experiments (*: P < 0.05, ***: P < 0.001) and rapamycin, were the most efficient, resulting in high Figure S3A), confirming that HCQ increases sensitivity autophagy levels (Fig. 2a). Following an IC50 concentra- to chemotherapy. The MDC test also showed a tion treatment, autophagy increased with all drugs in the dose-dependent increase of autophagosome numbers four cell lines, independently to MYCN amplification after treatment, enhanced by addition of HCQ, consistent (Fig. 2b). SK-N-SH and IGR-N91 cells incubated with with a late inhibition of autophagy (Fig. 4f and Additional vincristine for 24 h showed a moderate conversion of file 3: Figure S3B). LC3I to LC3II suggesting that cells undergo apoptosis. In addition, p62 expression decreased when cells were Autophagy inhibition decreases tumor progression in treated with this drug demonstrating the presence of vivo autophagy. This result was also confirmed by confocal To further investigate the effect of autophagy inhibition analysis where assessment of LC3 showed a constant in- on tumor progression, we compared tumor development crease in cytoplasmic autophagosome formation in cells in mice treated or not with HCQ, vincristine or a com- treated with vincristine (refer to Fig. 4c). This increased bination of HCQ and vincristine. At the end of the treat- of autophagy was correlated with an increase of acti- ment, the tumor volume was significantly lower in mice vated mTOR expression but only for the low concentra- treated with HCQ and HCQ combined to vincristine tion of vincristine (Fig. 2c). The induction of autophagy than in control (p < 0.05 and p < 0.01 respectively). The by chemotherapy was also found in in vivo experiments association of HCQ and vincristine is significantly more with strong LC3II positivity in tumors of NSG mice efficient than vincristine alone (p < 0.05). Interestingly, injected with SK-N-DZ and treated with cisplatin or HCQ used alone have a similar efficiency as vincristine IGR-N91 treated with vincristine (Fig. 2d). alone (Fig. 5). On another side, tumors were developed in 11/12 mice Inhibition of autophagy sensitizes NB cells to injected with sheGFP cells versus only 3/12 in mice kd chemotherapy injected with ATG5 cells (Additional file 4: Figure S4). Transfection efficiency with lentivirus-based short hair- kd pin RNA for ATG5 knockdown (ATG5 ) was confirmed Discussion kd by Western blot and by MDC (Fig. 3a and b). ATG5 Despite aggressive multimodal therapy, NB patients still cells were significantly more sensitive to vincristine, have a poor prognosis, which is partially explained by doxorubicin or cisplatin than sheGFP cells (Fig. 3c), sug- chemoresistance of the tumor cells. Furthermore, it has gesting that autophagy contributes to chemoresistance been reported that autophagy is a potential mechanism in NB. that promotes tumor cell survival and confers chemore- Treatment with HCQ induced an inhibition of the late sistance [29]. In our study, we observed that autophagy stage of autophagy which was shown by an increase of is present at basal levels to maintain homeostasis. LC3 the LC3II/LC3I ratio but no decrease of p62 in Western expression was not correlated to any clinicopathological blot (Fig. 4a), as well as an accumulation of autophago- data. However, autophagy was higher in Stage 4 tumors some detected by confocal microscopy (Fig. 4c and than 4S while apoptosis was lower in Stage 4 tumors Additional file 2: Figure S2) and an increase of MDC than stage 4S, which could explain the aggressive positivity (Fig. 4d). The inhibition of autophagy was not properties of Stage 4 tumors compared to 4S. Beclin 1 is correlated with increase of cell death (Fig. 4a and b). a factor of poor prognosis as it is highly expressed in tu- HCQ treatment does not modify the expression of mors from NB patients older than one year old. In other pmTOR but decreases phosphorylated AKT levels at cancers, such as human hypopharyngeal squamous cell high concentrations (Fig. 4a). carcinoma (HSCC), expression of Beclin 1 and LC3II The MTT test showed that cells treated with a com- correlates with poor prognosis [30]. Additionally, other bination of HCQ 30 μM and vincristine (0.001–100 μM) studies showed that autophagy predicts resistance to or doxorubicin (0.001–100 μM) were significantly more chemotherapy and survival in melanoma [31]. We also sensitive to chemotherapy than cells treated with vincris- observed a negative correlation between LC3II and tine or doxorubicin alone (Fig. 4e and Additional file 3: pAKT as well as pmTOR and between Beclin 1 and Belounis et al. BMC Cancer (2016) 16:891 Page 10 of 14 Fig. 4 (See legend on next page.) Belounis et al. BMC Cancer (2016) 16:891 Page 11 of 14 (See figure on previous page.) Fig. 4 HCQ sensitizes NB cells to chemotherapy by inhibition of autophagy. a IGR-N91 and SK-N-SH cells were treated with 15 to 60 μMofHCQ and analyzed for LC3B, p62, mTOR, p-mTOR, AKT, pAKT, Beclin 1 protein expression by immunoblotting. β-actin was used as a loading control. b Cell viability of SK-N-DZ, IGR-N91 and IGR-NB8 was measured using an MTT assay pre and post HCQ treatment (30 μM). c Confocal microscopy analysis was done on transfected IGR-N91 cells with GFP-LC3 not treated or treated 7 h with HCQ 30 μM, vincristine 1 μM or the combination of the two drugs. d Autophagy in SK-N-DZ, IGR-N91 and IGR-NB8 cells was measuredbyanMDC assaypre andpostHCQ treatment(30 μM). e SK-N-DZ cell viability was measured after vincristine or doxorubicin treatment combined or not with HCQ (30 μM). f The autophagic activity of SK-N-DZ cells was measured using a MDC agent after treatment with vincristine or doxorubicin, combined or not with HCQ (30 μM). Results are expressed as percentage of corresponding control and represent mean ± SEM of 4 independent experiments (*: P < 0.05, **: P < 0.01, ***: P < 0.001) pAKT, suggesting that autophagy activation occurs after increased autophagy comparing to other cell lines with inhibition of the AKT pathway since the inhibition of non-amplified MYCN. Temozolomide has been de- mTOR activates autophagy. scribed as an inducer of autophagy [35]. In the present To determine whether autophagy is activated after study, temozolomide doesn’t induce a dose-dependent chemotherapy treatment, three chemotherapeutic increase of autophagy, detectable by MDC and by agents, used clinically for NB treatment, were studied: Western blot. Differently, the present study showed that vincristine, doxorubicin and cisplatin. Our data demon- LY294002 was a dose dependent activator of autophagy. strate that the activation of autophagy by cisplatin and This drug increases autophagy by inhibiting PI3K I, an vincristine is dose-dependent. This is also observed with element of the PI3K/AKT/mTOR inhibitory pathway to doxorubicin except in one cell line (SK-N-FI). These autophagy [36]. Rapamycin induces autophagy by data were confirmed by in vivo study that show the acti- inhibiting mTOR [37]. In the present study, an increase vation of autophagy in NB tumors from mice receiving in autophagy detected by MDC was associated with the cisplatin or vincristine treatment compared to non- treatment by rapamycin in the four studied NB cell lines. treated tumors. In other cancers, doxorubicin is known It has been described that in the presence of rapamycin, as an inducer of the autophagosome formation in the dephosphorylation of many proteins activates the papillary thyroid cancer [32], whereas cisplatin induced transcription of ATG8 and ATG14 genes, known to be autophagy in esophageal squamous cell carcinoma cells associated with autophagy [13]. [33]. The MYCN gene amplification in several cell lines Beclin 1 has many cell functions, such as inducing used in our study could change the state of autophagy autophagosome formation and acting as a tumor sup- indeed several studies have showed that overexpression pressor [38]. It is a mediator of various cellular cascades, of CMYC strongly induces autophagy in rat 3Y1 fibro- including autophagy, apoptosis and cell differentiation blasts [33, 34]. Our data indicate that NB cell lines with [39]. The association of certain genes of the Bcl-2 family amplified MYCN (SK-N-DZ, IGR-N91) didn’t show with Beclin 1 inhibits the autophagic function by Fig. 5 Effect of autophagy inhibition by HCQ on tumors in vivo. Six NSG mice of each group received a s.c injection of 5x10 IGR-N91 cells. When tumors reached 100 mm , mice were administered vincristine (0.4 mg/kg/day), HCQ (60 mg/kg), an association of vincristine and HCQ or vehicle (saline) for 9 days. Thetumor size of thefourgroupsofmicewas compared during this period Belounis et al. BMC Cancer (2016) 16:891 Page 12 of 14 limiting its partnering with a class III PI3K and activates HCQ sensitizes cells to the conventional chemotherapy its apoptotic function instead [40]. It is the positive in NB treatment. Therefore, we propose that HCQ could regulation of Beclin 1 that induces the autophagic and be used as an adjuvant therapy in clinical trials to differentiation cascades [39]. Also, Beclin 1 loses its au- enhance the efficacy of chemotherapy in NB patients. tophagic abilities and induces apoptosis when there is an increased activity of caspase-9 [41]. This caspase has the Additional files property of cleaving Beclin 1 and creating a C-terminal fragment promoting pro-apoptotic activity [41]. Accord- Additional file 1: Figure S1. Control for immunogistochemistry. Normal mouse or rabbit IgG at the same concentration as the primary ing to some studies, doxorubicin activates caspase-9 and antibody were used as negative control (NC) and synaptophysin (1/100, positively regulates the expression of Beclin 1 [41]. Con- Polyclonal, SP11, Thermofisher Scientific) as positive control (PC). sequently, this particular NB drug induces apoptosis. (PDF 379 kb) Collectively, our data demonstrate that autophagy is acti- Additional file 2: Figure S2. GFP-LC3 transfection and confocal microscopy. The cell line IGR-N91 transfected with GFP-LC3 were not vated in response to chemotherapy in NB cells but has not treated (a), treated with vincristine alone (1 μM) (b), with HCQ (30 μM) answered if this autophagy is an autophagy-mediated cell alone (c) or with the association of the two drugs (d) and then analyzed death or autophagy-mediated cell survival (chemoresis- with confocal microscopy for 7 h. Treatment with HCQ induced an inhibition of the late stage of autophagy which was shown an tance). We investigated the effect of combining chemo- accumulation of autophagosome. (PDF 789 kb) therapy with autophagy inhibition. In the early stages of Additional file 3: Figure S3. HCQ sensitizes NB cells to chemotherapy carcinogenesis, autophagy acts as a primary tumor suppres- by inhibition of autophagy. A. N91-IGR or NB8-IGR cell viability was sor and inhibits tumor progression [21]. However, once measured after vincristine or doxorubicin treatment combined or not to HCQ 30 µM. Results are expressed as percentage of corresponding control tumor is established, autophagy tend to be a protective and represent mean ± SEM of 4 independent experiments. B. Autophagic mechanism used by tumors cells to overcome chemother- activity of N91-IGR or NB8-IGR was measured using MDC agent after apy and other metabolic stress. We used HCQ to target the increasing concentration of vincristine or doxorubicin treatment combined or not to HCQ 30 µM. Fluorescence was quantified by spectrophotometer. late stages of autophagy by blocking the fusion of autopha- (*: P <0.05), (**: P <0.01), (***: P < 0.001). (PDF 215 kb) gosomes with lysosomes [42]. Interestingly, mice treated Additional file 4: Figure S4. Effect of ATG5 knockdown xenografts on with HCQ alone have decreased tumors volume compared tumor growth. (11/12) NSG Mice developed xenograft tumor from s.c to mice treated with vincristine. This results support some injection of 5.10 sheGFP cells and only (3/12) from shATG5 cells (Table A). When tumor reached 200 mm3, mice were administered vincristine clinical trials that use HCQ alone to treat patients with (0.4 mg/kg/day) or vehicle (saline) for 5 days. Progression of tumor pancreatic cancer, breast cancer, renal cell carcinoma or volume was followed in each group. Each data point represents the chronic lymphocytic leukemia [23, 43]. In the present study, mean 6 ± SE tumor volume for sheGFP cells and 1 or 2 for shAtg5 cells. At day 6, Tumors size from shATG5 cells were reduced compared with HCQ was used at non-toxic concentrations that trigger tumors from eGFP (B). Also, tumors from shATG5 cells were more autophagy inhibition. Our data demonstrated that mice sensitive to vincristine comparing to control cells. Immunohistochemistry treated with a combination of vincristine and HCQ develop was performed on the sections of tumors developed in the mouse model. with LC3 antibody and showed a high expression of LC3 II in tumors with a significantly less extend than mice treated control cells and a very low in cells ATG5 knockdown as well as treated with vincristine alone. These data demonstrate, for the first (b, d) or not treated (a, c) (C). (PDF 259 kb) time, that inhibition of autophagy using HCQ sensitizes NB cells to classical chemotherapy supporting the idea that au- Abbreviations tophagy acts as a cytoprotective mechanism [44] and its in- ATG: Autophagy-related genes; Cis: Cisplatin; CQ: Chloroquine; DXR: Doxurubicin; HCQ: Hydroxycholoroquine; IHC: Immunohistochemistry; hibition may promote apoptosis in cancer cells [45]. Several LC3: Microtubule-associated protein light chain 3; studies and clinical trials have investigated autophagy MDC: Monodansylcadaverine; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5- inhibition using different pharmacological agents, usually in diphenyltetrazolium bromide; NB: Neuroblastoma; NSG: NOD scid gamma; shRNA: Short-hairpin RNA; TMA: Tissue microarray; VCR: Vincristine combination with chemotherapy, radiotherapy or other tar- geted anti-cancer therapies [46]. Some data demonstrated a Acknowledgements significant increase in long-term survival when the treat- This work was supported by grants from the Fondation Centre de ment includes CQ [47]. Reports from clinical trials indicate Cancérologie Charles-Bruneau, Canada and the Comité de Montbéliard ligue contre le cancer, France. We also thanks Dr Christian Beauséjour for providing that when CQ was added to conventional therapy, im- packaging plasmids. provement of mid-term survival for glioblastoma multiform patients was seen [48]. Taken together, these results Funding This study was supported by Fondation Centre de Cancérologie Charles-Bruneau, confirm that autophagy promotes chemoresistance and its Montreal, Canada and the Comité de Montbéliard ligue contre le cancer, France. inhibition sensitizes NB cells to chemotherapy. Availability of data and materials Conclusions All chemicals, reagents, drugs and plasmids were purchased. No chemicals or plasmids were formulated or manufactured in our laboratory, Our study demonstrates that autophagy is present in NB All data and results are already present in the manuscript and supporting tumors at a basal level and it is activated after chemo- files. There is thus no need to provide links to study data. The cell lines therapeutic that confers chemoresistance. The use of described in this study are available to investigators upon request. Belounis et al. BMC Cancer (2016) 16:891 Page 13 of 14 Authors’ contributions 15. Galluzzi L, et al. Guidelines for the use and interpretation of assays for monitoring All authors have read and approved the final text of the manuscript. AB cell death in higher eukaryotes. Cell Death Differ. 2009;16(8):1093–107. carried out the majority of the in vitro study (MTT, Immunoblot and 16. Miracco C, et al. Beclin 1 and LC3 autophagic gene expression in cutaneous autophagy assay), interpretation of the data, statistical analysis, and melanocytic lesions. Hum Pathol. 2010;41(4):503–12. manuscript drafting. CN conceived of the study, and participated in its 17. Fujiwara K, et al. Akt inhibitor shows anticancer and radiosensitizing effects in design and coordination and helped to draft the manuscript. RLG carried out malignant glioma cells by inducing autophagy. Int J Oncol. 2007;31(4):753–60. the in vivo study. MM carried out the production of lentiviral particles and 18. Petiot A, et al. Distinct classes of phosphatidylinositol 3'-kinases are involved transduction of NB cells. PT involved in drafting the manuscript or revising it in signaling pathways that control macroautophagy in HT-29 cells. J Biol critically for important intellectual content. MB and SC participated in the in Chem. 2000;275(2):992–8. vivo study. ÉH and GV contributed in results interpretation. HS carried out 19. Budanov AV, Karin M. p53 target genes sestrin1 and sestrin2 connect the interpretation of TMA, conceived of the study and participated in its genotoxic stress and mTOR signaling. Cell. 2008;134(3):451–60. design and coordination and helped to draft the manuscript. All authors 20. Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged read and approved the final manuscript. sword. Science. 2004;306(5698):990–5. 21. Turcotte S, Giaccia AJ. Targeting cancer cells through autophagy for Competing interests anticancer therapy. Curr Opin Cell Biol. 2010;22(2):246–51. The authors declare that they have no competing interests. 22. Paglin S, et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 2001;61(2):439–44. 23. Yang ZJ, et al. The role of autophagy in cancer: therapeutic implications. Consent for publication Mol Cancer Ther. 2011;10(9):1533–41. Not applicable. 24. Brodeur GM, et al. International criteria for diagnosis, staging, and response to treatment in patients with neuroblastoma. J Clin Oncol. 1988;6(12):1874–81. Ethics approval and consent to participate 25. Biederbick A, Kern HF, Elsasser HP. Monodansylcadaverine (MDC) is a specific in Written informed consent for the use of tissues for research was obtained vivo marker for autophagic vacuoles. Eur J Cell Biol. 1995;66(1):3–14. from patient guardians. The study was reviewed and approved by the 26. Burkhart CA, et al. Small-molecule multidrug resistance-associated protein 1 Research Ethics Board of the Sainte-Justine University Health Center. inhibitor reversan increases the therapeutic index of chemotherapy in This study employed animals and thus the general guidelines established by mouse models of neuroblastoma. Cancer Res. 2009;69(16):6573–80. the European Community (Directive 2010/63/UE) were followed, while the 27. Harned TM, et al. Sodium thiosulfate administered six hours after cisplatin experimental protocols were approved by the Institutional Committee for does not compromise antineuroblastoma activity. Clin Cancer Res. Ethics in Animal Research (CEEA26. Approval number: 2013–099). 2008;14(2):533–40. 28. Das B, et al. Squalene selectively protects mouse bone marrow progenitors Author details against cisplatin and carboplatin-induced cytotoxicity in vivo without Research centre of the Sainte Justine university hospital, Montreal, QC, protecting tumor growth. Neoplasia. 2008;10(10):1105–19. Canada. Department of pathology and cellular biology, Université de 29. Sui X, et al. Autophagy and chemotherapy resistance: a promising Montréal, Montreal, QC, Canada. Department of biochemistry, CHU Sainte therapeutic target for cancer treatment. Cell Death Dis. 2013;4:e838. Justine, Montreal, QC, Canada. Division of paediatric oncology, CHU Sainte 30. Wang J, et al. Aberrant expression of Beclin-1 and LC3 correlates with poor Justine, Montreal, QC, Canada. Department of surgery, CHU Sainte Justine, prognosis of human hypopharyngeal squamous cell carcinoma. PLoS One. 3175 Montreal, QC, Canada. Department of paediatric oncology, Institut 2013;8(7):e69038. Gustave Roussy, Villejuif, France. Department of pathology and cytogenetic, 31. Ma XH, et al. Measurements of tumor cell autophagy predict invasiveness, CHU Sainte Justine, Montreal, QC, Canada. resistance to chemotherapy, and survival in melanoma. Clin Cancer Res. 2011;17(10):3478–89. Received: 4 April 2016 Accepted: 27 October 2016 32. Lin CI, et al. Autophagy: a new target for advanced papillary thyroid cancer therapy. Surgery. 2009;146(6):1208–14. 33. Liu D, et al. Inhibition of autophagy by 3-MA potentiates cisplatin-induced References apoptosis in esophageal squamous cell carcinoma cells. Med Oncol. 1. Schwab M, et al. Neuroblastoma: biology and molecular and chromosomal 2011;28(1):105–11. pathology. Lancet Oncol. 2003;4(8):472–80. 34. Tsuneoka M, et al. c-myc induces autophagy in rat 3Y1 fibroblast cells. Cell 2. Canadian Cancer Society's Steering Commitee. Canadian Cancer Statistics Struct Funct. 2003;28(3):195–204. 2009. 2009. 35. Kanzawa T, et al. Role of autophagy in temozolomide-induced cytotoxicity 3. Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat for malignant glioma cells. Cell Death Differ. 2004;11(4):448–57. Rev Cancer. 2003;3(3):203–16. 36. Xing C, et al. Class I phosphatidylinositol 3-kinase inhibitor LY294002 activates 4. Maris JM. The biologic basis for neuroblastoma heterogeneity and risk autophagy and induces apoptosis through p53 pathway in gastric cancer cell stratification. Curr Opin Pediatr. 2005;17(1):7–13. line SGC7901. Acta Biochim Biophys Sin (Shanghai). 2008;40(3):194–201. 5. Park JR, Eggert A, Caron H. Neuroblastoma: biology, prognosis, and 37. Chang YY, et al. Nutrient-dependent regulation of autophagy through the treatment. Pediatr Clin North Am. 2008;55(1):97–120. x. target of rapamycin pathway. Biochem Soc Trans. 2009;37(Pt 1):232–6. 6. Matthay KK, et al. Treatment of high-risk neuroblastoma with intensive 38. Pirtoli L, et al. The prognostic role of Beclin 1 protein expression in high- chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13- grade gliomas. Autophagy. 2009;5(7):930–6. cis-retinoic acid. Children's Cancer Group. N Engl J Med. 1999;341(16):1165–73. 39. Wang J. Beclin 1 bridges autophagy, apoptosis and differentiation. 7. Maris JM, et al. Neuroblastoma. Lancet. 2007;369(9579):2106–20. Autophagy. 2008;4(7):947–8. 8. He C, Klionsky DJ. Regulation mechanisms and signaling pathways of 40. Wei Y, et al. JNK1-mediated phosphorylation of Bcl-2 regulates starvation- autophagy. Annu Rev Genet. 2009;43:67–93. induced autophagy. Mol Cell. 2008;30(6):678–88. 9. Klionsky DJ. Autophagy: from phenomenology to molecular understanding 41. Furuya D, et al. Beclin 1 augmented cis-diamminedichloroplatinum induced in less than a decade. Nat Rev Mol Cell Biol. 2007;8(11):931–7. apoptosis via enhancing caspase-9 activity. Exp Cell Res. 2005;307(1):26–40. 10. Reggiori F, Klionsky DJ. Autophagosomes: biogenesis from scratch? Curr 42. Ben-Zvi I, et al. Hydroxychloroquine: from malaria to autoimmunity. Clin Rev Opin Cell Biol. 2005;17(4):415–22. Allergy Immunol. 2012;42(2):145–53. 11. Chen D, et al. A mammalian autophagosome maturation mechanism mediated by TECPR1 and the Atg12-Atg5 conjugate. Mol Cell. 2012;45(5):629–41. 43. Lagneaux L, et al. Early induction of apoptosis in B-chronic lymphocytic 12. Eskelinen EL, Saftig P. Autophagy: a lysosomal degradation pathway with a leukaemia cells by hydroxychloroquine: activation of caspase-3 and no central role in health and disease. Biochim Biophys Acta. 2009;1793(4):664–73. protection by survival factors. Br J Haematol. 2001;112(2):344–52. 13. Yang YP, et al. Molecular mechanism and regulation of autophagy. Acta 44. Shen S, et al. Association and dissociation of autophagy, apoptosis and Pharmacol Sin. 2005;26(12):1421–34. necrosis by systematic chemical study. Oncogene. 2011;30(45):4544–56. 14. Yu L, Strandberg L, Lenardo MJ. The selectivity of autophagy and its role in 45. Maiuri MC, et al. Self-eating and self-killing: crosstalk between autophagy cell death and survival. Autophagy. 2008;4(5):567–73. and apoptosis. Nat Rev Mol Cell Biol. 2007;8(9):741–52. Belounis et al. BMC Cancer (2016) 16:891 Page 14 of 14 46. White E, DiPaola RS. The double-edged sword of autophagy modulation in cancer. Clin Cancer Res. 2009;15(17):5308–16. 47. Liang X, et al. Inhibiting systemic autophagy during interleukin 2 immunotherapy promotes long-term tumor regression. Cancer Res. 2012;72(11):2791–801. 48. Sotelo J, Briceno E, Lopez-Gonzalez MA. Adding chloroquine to conventional treatment for glioblastoma multiforme: a randomized, double- blind, placebo-controlled trial. Ann Intern Med. 2006;144(5):337–43. 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Pubmed Central
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© The Author(s). 2016
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1471-2407
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1471-2407
DOI
10.1186/s12885-016-2906-9
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Abstract

Background: Neuroblastoma (NB) is a frequent pediatric tumor characterized by a poor prognosis where a majority of tumors progress despite intensive multimodality treatments. Autophagy, a self-degradative process in cells, could be induced by chemotherapy and be associated with chemoresistance. The aim of this study was to determine whether: 1) autophagy is present in NB, 2) chemotherapy modified its levels, and 3) its inhibition decreased chemoresistance. Methods: Immunohistochemical stainings were performed on samples from 184 NB patients in order to verify the expression of LC3B, a specific marker for autophagy, and Beclin 1, a positive regulator of autophagy. In addition, we performed an in vitro study with six NB cell lines and six drugs (vincristine, doxorubicin, cisplatin temozolomide, LY294002 and syrolimus). Inhibition of autophagy was performed using ATG5 knockdown cells or hydroxychloroquine (HCQ). Cell survival was measured using the MTT cell proliferation assay. Autophagy was detected by monodansylcadaverine, confocal microscopy and Western blot. In vivo study with tumor xenografts in NSG mice was performed. Results: Our results have indicated that autophagy was present at low levels in NB and was not a prognostic factor, while Beclin 1 was highly expressed in children with poor NB prognosis. However, autophagy levels increased after chemotherapy in vitro and in vivo. Tumor progression was significantly decreased in mice treated with a combination of HCQ and vincristine. Conclusions: Taken together, autophagy is present in NB, induced by chemotherapy and associated with chemoresistance, which is significantly reduced by its inhibition. Therefore, targeting autophagy represents a very attractive approach to develop new therapeutic strategies in NB. Keywords: Neuroblastoma, Autophagy, Chemoresistance, Hydroxychloroquine Background resistance to high-dose chemotherapy indicate that NB is Neuroblastoma (NB) is the most common and deadly ex- specifically associated with chemoresistance [7]. tracranial solid tumor in children [1, 2]. Survival of children Autophagy is a ubiquitous self-degradation process that older than one year of age with advanced NB is poor (only involves the degradation and recycling of cellular cytoplas- 34%), despite aggressive treatments [3, 4]. High-dose mic constituents through the lysosomal pathway. Damaged chemotherapy with autologous hematopoietic stem cell or misfolded proteins or organelles are first sequestered in transplantation significantlyimprovesthe prognosisof double-membrane vesicles, known as autophagosomes, metastatic NB [5, 6], but this treatment carries with it a before fusing with lysosomes, where their contents are de- high risk of adverse effects [4]. Poor global survival and graded by lysosomal proteases [8–10]. Autophagy is a com- plex and multistep process involving the autophagy-related proteins (ATG) [8]. ATG5 is a protein involved in the early * Correspondence: hsartelet@chu-grenoble.fr Research centre of the Sainte Justine university hospital, Montreal, QC, stages of autophagosome formation and plays an essential Canada role in the maturation of autophagosomes [11], with assist- Department of pathology and cellular biology, Université de Montréal, ance from LC3 [8]. Low levels of autophagic activity are Montreal, QC, Canada Full list of author information is available at the end of the article © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Belounis et al. BMC Cancer (2016) 16:891 Page 2 of 14 commonly observed under normal conditions, presumably System (INSS) [24]. Treatment was assigned according preserving normal cellular homeostasis [12, 13]. Prolonged to the risk group on the basis of the patient’s age at time autophagy may result in type 2 (autophagic) programmed of diagnosis, the INSS stage, the histoprognosis, the cell death [12, 14]. The activation of autophagy is measured ploidy and MYCN amplification status (v-myc avian by the ratio between LC3II on the autophagosome mem- myelocytomatosis viral oncogene neuroblastoma derived brane and LC3I in the cytoplasm which can be detected by homolog). With formalin-fixed and paraffin-embedded Western-blot [15] or by immunohistochemistry [16]. Beclin samples, a tissue microarray (TMA) was constructed 1 is also a marker and a positive regulator of autophagy. using four representative NB tumor tissue cylinders with The regulation of autophagy by the PI3K/AKT pathway a 0.6 mm diameter. TMA blocks contained not only 184 is very complex. Recently, an AKT inhibitor was reported primary tumors but also 47 paired metastases (42 lymph to induce autophagy with a radiosensitizing effect [17]. Au- nodes and 5 hepatic metastases). Among the 184 tophagy is regulated by both class I and III PI3K pathways tumors, 19 tumors were tested by Western blot, proteins [18, 19]. mTOR serves as a metabolic sensor that coordi- coming from the lysate of frozen samples. nates cross-talk between nutrient availability and autophagy [19]. On the other hand, class III PI3K in conjunction with Immunohistochemistry Beclin 1 positively regulates autophagy [18, 19]. Immunohistochemistry was performed on the sections of In cancer, autophagy plays a dual role by either activating the TMA blocks or of tumors developed in the mouse cell death and inhibiting tumor progression or promoting model. The Ultraview Universal DAB detection kit cell survival [20]. In the early stages of carcinogenesis, (Ventana, Ventana medical system, Tuscon, AR) was used. autophagy acts as a primary tumor suppressor and inhibits Antibodies against phospho-AKT (1/100, S473-r, Santa tumor progression [21]. However, autophagy can also Cruz biotechnology, CA), phospho-mTOR (1/100, 49 F9, confer tumor cells the ability to resist to ionizing radiation Cell Signaling, CA), LC3B (1/1000, ab51520 abcam, [22] as well as to chemotherapy [23]. Cambridge UK) or Beclin 1 (1/250, ab55878 abcam) were The observation of increased cell survival associated with applied for 30 min. DAB was used as a chromogen and higher autophagy activity following therapy has led to the hematoxylin as a counterstain. Normal mouse or rabbit development of strategies combining autophagy inhibitors IgG at the same concentration as the primary antibody to current anticancer treatments. In this context, chloro- were used as negative control and synaptophysin (1/100, quine (CQ) or its derivate hydroxychloroquine (HCQ), sen- Polyclonal, SP11, Thermofisher Scientific) as positive sitizes tumor cells to anticancer therapies. Indeed, CQ and control (Additional file 1: Figure S1). Two investigators HCQ block the processing and maturation of autophagy blinded for clinical data independently evaluated vacuoles (autophagolysosomes) by inhibiting lysosomal ac- immunostaining in samples containing more than 100 NB tivity [23]. Some data suggest that autophagy inhibition and cells. Immunostaining scores were established by a semi- autophagosome accumulation both contribute to the quantitative optical analysis assessing the percentage of accelerated cell death induced by HCQ [23]. positive cells in each sample: 0 = all cells negative, 1 + = 1 The aim of the present study is to demonstrate the to 25%, 2 + = 26 to 50%, 3 + = 51 to 75% and 4+ more than presence of autophagy in NB, its activation by chemo- 75% of positive tumoral cells. therapy and its correlation with chemoresistance. TUNEL Methods On the sections of TMA, a terminal deoxynucleotidyl Study design and patients transferase-mediated dUTP nick end-labeling (TUNEL) Study cases were selected upon the following inclusion assay (In situ cell death detection kit, POD (Roche)) was criteria; 1) a diagnosis of NB had been made between used to identify double-stranded DNA fragmentation, char- July 1988 and March 2008, 2) human subject research acteristic of DNA degradation due to apoptosis. Briefly, tis- (tissue samples) was approved by the Research Ethics sue slides were deparaffinized. The slides were then treated Board of the Sainte-Justine University Health Center. with 0.1% of Triton X-100 (Sigma, X-100). The slides were Written consent has been obtained from patient then incubated with terminal deoxynucleotidyl transferase guardians, and 3) adequate specimen material has been followed by peroxidase-conjugated anti-digoxigenin anti- collected for study purposes. 184 patients with NB were body. Finally, the slides were stained with DAB. Methyl included in our study. The patients were treated and green was performed as the counter-stain. Slides were followed up at Sainte-Justine University Health Center scanned using a customized, computer-controlled micro- (Montreal, Canada). Thirty-one out of 184 total NB scope (Axio Imager M1; Zeiss, Oberkochen, Germany). cases were identified from routine provincial (Quebec, The percentage of positive neuroblasts for TUNEL was also Canada) mass screening efforts. Tumors were classified calculated by dividing the number of stained nuclei by the according to the International Neuroblastoma Staging total numbers of neuroblasts and multiplying by 100. Belounis et al. BMC Cancer (2016) 16:891 Page 3 of 14 Table 1 Patients clinical data Variable LC3B Beclin-1 No (%) No % Mean p No % Mean p Intensity Intensity All patients 184 148 80 0.83 153 83 1.03 Age Median (Range), months 26 (0–151) <365 days 88 (48) 73 83 0.92 70 80 0.83 ≥365 days 96 (52) 74 77 0.75 ns 84 87.5 1.25 <0.01 Stage 1 53 (29) 47 88 0.6 ns 48 90 1.30 ns 2 35 (19) 25 71 0.64 27 77 0.8 3 23 (12) 21 91 0.94 20 86 1.14 4 58 (32) 52 89 1.12 47 81 0.97 4S 15 (8) 11 73 0.34 0.02 11 73 0.72 ns MYCN status Amplified 14 (10) 13 87 0.81 11 73 0.87 Non Amplified 127 (90) 103 81 0.50 ns 105 82 0.43 ns Unknown 43 Type of NB Standard 149 (81) 133 89 0.96 126 84 1.19 Mass screening 35 (19) 29 82 0.8 ns 27 77 0.46 <0.001 Type of samples Primary tumors 184 148 80 0.83 153 83 1.03 Metastases 47 41 87 0.78 ns 37 79 0.69 <0.01 Cell lines medium for 2 days before selection for stable expression of Six NB cell lines were studied: SK-N-DZ (ATCC, Manassas, theshRNA by growinginculture mediacontaining VA), SK-N-SH (ATCC), SK-N-FI (ATCC), IGR-N91 and puromycin (5 μg/mL)for at least2weeks. IGR-NB8 cells from Gustave Roussy Institute (Villejuif, France), and NB-10 (St. Jude Children’sHospital, Memphis, GFP-LC3 transfection and confocal microscopy TN). MYCN amplification is present in NB-10, SK-N-DZ The cell line IGR-N91 was transfected with GFP-LC3 and IGR-N91 cells. The cells were cultured in Dulbecco’s (Millipore’s LentiBrite TM GFP-LC3 lentiviral Biosensor) modified Eagle medium (DMEM), 10% fetal bovine serum for monitoring autophagosome formation. IGR-N91 cells (FBS) and 1% penicillin-streptomycin at 37 °C in a 5% CO were seeded at 4 × 10 /well into eight-well chamber slides atmosphere. (Thermo Scientific, Rochester, NY) to achieve 70% confluence. After 24 h, the cells were transfected with Knockdown of ATG5 expression by lentivirus-delivered lentivirus containing a version of GFP-LC3 at 37 °C, 5% shRNA CO for 24 h. At 48 h post-transfection, the medium was TRC Lentiviral Human ATG5 and eGFP shRNA vectors changed to DMEM, 1% fetal bovine serum (FBS) and 1% (ATG5: accession #NM_004849, eGFP: accession # penicillin-streptomycin, the cells were washed three times RHS4459) were purchased from Open Biosystems, with PBS and visualized with the Ultraview Vox Confocal Rockford, IL. Lentiviral vectors were produced using HEK Imaging System (Perkin Elmer). The autophagosome 293 T cells by PEG (polythylenimine linear, Polysciences volume in transfected cells was evaluated with Imaris 7.7.2 inc) transfection of ATG5 or eGFP shRNA plasmid software (Oxford Instruments Company). together with the third-generation packaging plasmids pMDL, pRev and pV-SVG (Open Biosystems). To generate Cell proliferation assay human ATG5-knockdown cells, IGR-N91 cells were Cell viability was determined by the MTT test. Cells transduced with lentivirus expressing shATG5 or sheGFP were plated in a 96-well culture plate at a density of for control. Transduced cells were cultured in fresh 5x10 /well over night. The cells were treated with Belounis et al. BMC Cancer (2016) 16:891 Page 4 of 14 Fig. 1 (See legend on next page.) Belounis et al. BMC Cancer (2016) 16:891 Page 5 of 14 (See figure on previous page.) Fig. 1 Evaluation of the level and the regulation of autophagy in NB and its correlation with apoptosis. A Autophagy was evaluated in TMA sections from NB tumor samples by immunohistochemistry using antibodies anti-LC3B (A.a,b) or anti-Beclin 1 (A.c). The expression levels of pmTOR (A.e) and pAKT (A.f), two autophagy-regulating proteins, were also studied on the same samples. Apoptosis was tested by TUNEL (A.d). Scale bars: 100 μm. B Immunoblotting analysis was performed on protein lysates from different frozen matched tumor samples with antibodies against either LC3B or Beclin 1 for autophagy and against cleaved caspase-3 for apoptosis. C The expression of LC3II and TUNEL positivity were semi-quantified according to the stage of tumors. D The ratio LC3II/LC3I and cleaved caspase-3 expression were evaluated by densitometry using tumor samples various conditions for 24 h then proliferation was mea- or with the association of the two drugs and then analyzed sured by the (3-[4,5dimethyl-2-thiazolyl]-2,5-diphenyl- with confocal microscopy for 7 h. 2H-tetrazolium bromide) MTT cell proliferation assay (Cell Titer 96 Non-Radioactive Cell proliferation Assay, Western Blot Promega) according to manufacturer’s instructions. Protein extracts were prepared from frozen tumor Absorbance was measured at 570 nm using the Spectra tissues of 19 patients and cultured cell lines. Cell lysis Max 190 microplate spectrophotometer (Molecular buffer with protease inhibitor cocktail (10 mM Tris–HCl Devices, Sunnyvale, CA). Assays were performed in pH 7.4, 150 nM NaCl, 1 mM EDTA, 1 mM EGTA, 1% triplicate. Relative cell viability (percent of control) was Triton X-100, 0.5% NP-40, 1 mM sodium orthovanadate, calculated using the equation: (mean OD of treated 1 mM sodium fluoride) was added to each sample. Equal cells/mean OD of control cells) × 100. For each time amounts of protein (20 μg) from cell lysates were solubi- point, the treated cells were compared with control cells lized in Laemmli sample buffer, boiled for 5 min, sepa- that had been treated with vehicle only. rated by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes, blocked 1 h at room Monodansylcadaverine (MDC) test temperature with TBS buffer (20 mM Tris–HCl, pH 7.4, To correlate cell survival with the presence of autopha- 150 mM NaCl) containing 3% bovine serum albumin, gic vacuoles, cells were incubated with the autofluores- and incubated with primary antibody overnight at 4 °C. cent agent monodansylcadaverine (MDC) at 0.05 mM in Immunoreactive bands were revealed following 1 h PBS (Sigma) for 10 min at 37 °C. MDC has been incubation with horseradish peroxidase-conjugated reported to specifically label autophagic vacuoles [25]. anti-rabbit or anti-mouse antibodies, and the signals MDC was then replaced with 200 μl of PBS and finally were visualized with an enhanced chemiluminescence replaced by 100 μl of Tris-Triton (Tris 10 mM pH 8.0, (ECL) detection system (PerkinElmer, Waltham, MA). 0.1% Triton X-100). Fluorescence was measured with The primary antibodies used for this study were: β-actin EnVision 2104 multilabel reader (Perkin Elmer) with an (13E5; diluted 1:5000, Cell Signaling, Danvers, MA), emission filter of 525 nm at 380 nm. mouse polyclonal anti-human PARP-1 (Ab-2; 1:600; Calbiochem, Billerica, MA), rabbit polyclonal anti- Cell treatments human Beclin 1 (1/1000, ab55878 abcam, Cambridge The MTT and MDC tests were performed on cell lines UK), rabbit polyclonal anti-human SQSTM1/p62, LC3B with or without transfection of ATG5 shRNA vector and and ATG5, rabbit monoclonal anti-human Cleaved treated with different concentrations of drugs for 24 h at Caspase-3 (Asp175), mTOR (7C10), phospho-mTOR 37 °C: temozolomide (Schering Plough inc, 0.1–1000 μM), (Ser2448, D9C2), Akt and phospho-Akt (Ser473), (all vincristine (Mayne Pharma USA inc, Paramus NJ, diluted 1:1000, Cell Signaling, Danvers, MA). A densi- 1–10000 nM), doxorubicin (Pfizer Inc Kirkland, Canada, tometry analysis with the Kodak ID 3.6 software was 0.005–50 μM) and cisplatin (Mayne Pharma USA inc, used to calculate the relative expression of Cleaved Paramus NJ; 0.015–150 μg/ml), LY294002, specific Caspase-3 and the ratio between LC3B-II and LC3B-I. inhibitor of AKT (Calbiochem Darmstadt, Germany, 0.05–500 μM) and rapamycin, specific inhibitor of mTOR Animal experiments (Pfizer Inc Kirkland, Canada 0.1–1000 nM). Finally, cells NOD/LtSz-scid/IL-2Rgamma null mice (NSG) were pur- were treated with varying concentrations of cisplatin chased from Jackson Laboratory (Bar Harbor, ME, USA). (0.15–75 μg/mL) and doxorubicin (0.005–5 μg/mL) in the Animal experiments were approved by the CEEA26 presence and absence of HCQ (30 μM) for 48 h. The com- Ethics Committee (approval number: 2013–099) and pound concentrations resulting in 50% inhibition of cell carried-out under the conditions established by the viability (IC ) were determined using GraphPad Prism 6 European Community (Directive 2010/63/UE). All mice software. The cell line IGR-N91 transfected with GFP-LC3 were housed in ventilated cages under standard condi- were treated with vincristine (1 μM), with HCQ (30 μM) tions of controlled temperature and humidity, and Belounis et al. BMC Cancer (2016) 16:891 Page 6 of 14 Fig. 2 (See legend on next page.) Belounis et al. BMC Cancer (2016) 16:891 Page 7 of 14 (See figure on previous page.) Fig. 2 Chemotherapy induces autophagy in vitro and in vivo. The effect of chemotherapeutic agents on the autophagy of NB cell lines and tissues was analyzed. a NB-10 cells were treated with increasing concentrations of temozolomide, rapamycin, cisplatin, vincristine, doxorubicin or LY294002 for 24 h followed by MTT and MDC assays to measure cell viability and autophagy levels, respectively. Results are expressed as percentage of the corresponding control and represent mean ± SEM of 3 independent experiments (***: P < 0.001). b Cell viability and MDC specific autophagy were measured after IC50 treatment of different NB cell lines with chemotherapeutic agents. Results are expressed as percentage of the corresponding control and represent mean ± SEM of 3 independent experiments (***: P < 0.001). c Immunobloting analyses were performed on protein lysates from SK-N-SH and IGR-N91 cells treated with 0.1 to 10 μM of vincristine. Anti PARP-1, anti-LC3B, anti-p62, anti-mTOR, anti-pmTOR antibodies were used. β-actin was used as a loading control. d Tumor xenografts developed from SK-N-DZ or IGR-N91 cells were treated with cisplatin or vincristine. Slides were immunostained with anti-LC3B antibody. Scale bars: 100 μm exposed to a 12-hourly light/dark cycle. They were Autophagy and neuroblastoma provided with standard diet and water ad libitum. First, The analysis of LC3 immunostaining clearly showed two NSG were established in 6 to 8 week-old mice by sub- types of positivity: uniform cytoplasmic staining, corre- cutaneous injection of 5 × 10 IGR-N91 in the left flank. sponding to LC3I (Fig. 1Aa) and a punctate cytoplasmic Mice were monitored twice a week for tumor growth. LC3-staining pattern specific to LC3II and therefore to Clinical monitoring of mice (weight, vital signs, behavior autophagy (Fig. 1Ab). Furthermore, autophagy was and abdominal palpation) were carried out once a week. frequent in NB (80% of the tumors) but with a very low When the palpable tumor reached 100 mm (measured intensity (mean intensity 0.83). These data were con- with calipers), 6 mice/group received a dose of vincris- firmed by Western blot analysis where LC3II expression tine (0.4 mg/kg) [26] or HCQ (60 mg/kg) or an associ- was found in 12 out of 19 tumors (63%) (Fig. 1B). The ation of vincristine and HCQ at the same concentration cytoplasmic expression of Beclin 1 was also expressed in was injected daily for 9 consecutive days. Six control a majority of NB (83%) but with a low intensity (mean mice received (100 μL) of saline solution per day. Mice intensity 1.03) (Fig. 1Ac, Table 1). These data were were euthanized at the onset of clinical signs or when confirmed by Western blot analysis showing Beclin 1 bearing tumors of 1000 mm or at the end of the expression in 17 out of 19 tumors (90%) (Fig. 1B). treatment. The LC3II expression was significantly higher in Stage Secondly, five millions of SK-N-DZ cells (100 μL) were 4 (mean intensity 1.12) than in Stage 4S (mean intensity injected s.c. in the left flank. Mice were treated with 0.34) (Table 1, p = 0.02), confirmed by Western blot, vincristine (0.4 mg/kg) or cisplatin (Mayne Pharma, where LC3II was undetectable in specimen from Stage Canada) i.p. at 8 mg/kg for 4 days [27, 28]. Mice 4S patients. Interestingly, the level of apoptosis detected receiving PBS at the same time were used as a negative by TUNEL (Fig. 1Ad) and by cleavage of caspase-3 was control. In all in vivo experiments, mice were euthanized inversely correlated with autophagy, particularly in 10 days after the end of treatment or when tumor size Stages 4 and 4S (Fig. 1C and D). However, expression reached 1000 mm . Tumors were collected and levels of Beclin 1 were significantly higher in patients immunohistochemistry with LC3B antibody was older than one year at diagnosis compared to younger performed. patients (p < 0.001) (Table 1), in primary tumors than in metastasis (p < 0.01) and in standard NBs than in tumors coming from mass screening (p < 0.001), demonstrating Statistical analysis that Beclin 1 is highly expressed in NB with poor Association tests were performed with the use of Fisher’s prognosis. Among the major proteins present in the exact test. Spearman correlation values (rho) were used AKT pathway, there was a significant negative correl- to compare the expression between LC3-II and pmTOR, ation between LC3II and pmTOR (Fig. 1Ae) (rho = pAKT and between Beclin 1 and pAKT. Statistical ana- −0.22, p < 0.01) as well as pAKT (rho = −0.22, p < 0.01) lyses were performed using GraphPad Prism 6 software. and between Beclin 1 and pAKT (Fig. 1Af) (rho = −0.13, P values of less than 0.05 were considered to indicate p < 0.01), suggesting that autophagy is activated through statistical significance. AKT pathway inhibition. Results Chemotherapy induces autophagy in neuroblastoma cells Characteristics of patients The formation of autophagosomes revealed by MDC Clinicopathological characteristics of the 184 patients indicates a significant increase of autophagy following and tumors are detailed in (Table 1). These patients had treatment with increasing concentrations of cisplatin, a median follow-up period of 67 months (range vincristine and doxorubicin (Fig. 2a). Temozolomide newborn-174 months) and a median age at the time of didn’t induce high levels of autophagy even at high con- diagnosis to be 26 months (range newborn-151 months). centrations. AKT pathway inhibitors, such as LY294002 Belounis et al. BMC Cancer (2016) 16:891 Page 8 of 14 Fig. 3 (See legend on next page.) Belounis et al. BMC Cancer (2016) 16:891 Page 9 of 14 (See figure on previous page.) Fig. 3 Inhibition of autophagy sensitizes NB cells to chemotherapy. IGR-N91 cells were transduced with ATG5-shRNA lentivirus to knockdown ATG5 gene expression and thereby inhibit autophagy or with eGFP-shRNA, used as a control. a Protein immunoblotting using anti-ATG5 and anti-LC3B antibodies was performed to demonstrate ATG5 gene knockdown and LC3 expression on IGR-N91-transduced cells or not (WT). β-actin was used as a loading control. kd b A MDC test for autophagy was performed on ATG5 and control cells after treatment with increasing concentrations of vincristine. c Cell viability of kd ATG5 and control cells was measured with a MTT assay following treatment with increasing concentrations of vincristine, doxorubicin or cisplatin. Results are expressed as percentage of corresponding control and represent mean ± SEM of 4 independent experiments (*: P < 0.05, ***: P < 0.001) and rapamycin, were the most efficient, resulting in high Figure S3A), confirming that HCQ increases sensitivity autophagy levels (Fig. 2a). Following an IC50 concentra- to chemotherapy. The MDC test also showed a tion treatment, autophagy increased with all drugs in the dose-dependent increase of autophagosome numbers four cell lines, independently to MYCN amplification after treatment, enhanced by addition of HCQ, consistent (Fig. 2b). SK-N-SH and IGR-N91 cells incubated with with a late inhibition of autophagy (Fig. 4f and Additional vincristine for 24 h showed a moderate conversion of file 3: Figure S3B). LC3I to LC3II suggesting that cells undergo apoptosis. In addition, p62 expression decreased when cells were Autophagy inhibition decreases tumor progression in treated with this drug demonstrating the presence of vivo autophagy. This result was also confirmed by confocal To further investigate the effect of autophagy inhibition analysis where assessment of LC3 showed a constant in- on tumor progression, we compared tumor development crease in cytoplasmic autophagosome formation in cells in mice treated or not with HCQ, vincristine or a com- treated with vincristine (refer to Fig. 4c). This increased bination of HCQ and vincristine. At the end of the treat- of autophagy was correlated with an increase of acti- ment, the tumor volume was significantly lower in mice vated mTOR expression but only for the low concentra- treated with HCQ and HCQ combined to vincristine tion of vincristine (Fig. 2c). The induction of autophagy than in control (p < 0.05 and p < 0.01 respectively). The by chemotherapy was also found in in vivo experiments association of HCQ and vincristine is significantly more with strong LC3II positivity in tumors of NSG mice efficient than vincristine alone (p < 0.05). Interestingly, injected with SK-N-DZ and treated with cisplatin or HCQ used alone have a similar efficiency as vincristine IGR-N91 treated with vincristine (Fig. 2d). alone (Fig. 5). On another side, tumors were developed in 11/12 mice Inhibition of autophagy sensitizes NB cells to injected with sheGFP cells versus only 3/12 in mice kd chemotherapy injected with ATG5 cells (Additional file 4: Figure S4). Transfection efficiency with lentivirus-based short hair- kd pin RNA for ATG5 knockdown (ATG5 ) was confirmed Discussion kd by Western blot and by MDC (Fig. 3a and b). ATG5 Despite aggressive multimodal therapy, NB patients still cells were significantly more sensitive to vincristine, have a poor prognosis, which is partially explained by doxorubicin or cisplatin than sheGFP cells (Fig. 3c), sug- chemoresistance of the tumor cells. Furthermore, it has gesting that autophagy contributes to chemoresistance been reported that autophagy is a potential mechanism in NB. that promotes tumor cell survival and confers chemore- Treatment with HCQ induced an inhibition of the late sistance [29]. In our study, we observed that autophagy stage of autophagy which was shown by an increase of is present at basal levels to maintain homeostasis. LC3 the LC3II/LC3I ratio but no decrease of p62 in Western expression was not correlated to any clinicopathological blot (Fig. 4a), as well as an accumulation of autophago- data. However, autophagy was higher in Stage 4 tumors some detected by confocal microscopy (Fig. 4c and than 4S while apoptosis was lower in Stage 4 tumors Additional file 2: Figure S2) and an increase of MDC than stage 4S, which could explain the aggressive positivity (Fig. 4d). The inhibition of autophagy was not properties of Stage 4 tumors compared to 4S. Beclin 1 is correlated with increase of cell death (Fig. 4a and b). a factor of poor prognosis as it is highly expressed in tu- HCQ treatment does not modify the expression of mors from NB patients older than one year old. In other pmTOR but decreases phosphorylated AKT levels at cancers, such as human hypopharyngeal squamous cell high concentrations (Fig. 4a). carcinoma (HSCC), expression of Beclin 1 and LC3II The MTT test showed that cells treated with a com- correlates with poor prognosis [30]. Additionally, other bination of HCQ 30 μM and vincristine (0.001–100 μM) studies showed that autophagy predicts resistance to or doxorubicin (0.001–100 μM) were significantly more chemotherapy and survival in melanoma [31]. We also sensitive to chemotherapy than cells treated with vincris- observed a negative correlation between LC3II and tine or doxorubicin alone (Fig. 4e and Additional file 3: pAKT as well as pmTOR and between Beclin 1 and Belounis et al. BMC Cancer (2016) 16:891 Page 10 of 14 Fig. 4 (See legend on next page.) Belounis et al. BMC Cancer (2016) 16:891 Page 11 of 14 (See figure on previous page.) Fig. 4 HCQ sensitizes NB cells to chemotherapy by inhibition of autophagy. a IGR-N91 and SK-N-SH cells were treated with 15 to 60 μMofHCQ and analyzed for LC3B, p62, mTOR, p-mTOR, AKT, pAKT, Beclin 1 protein expression by immunoblotting. β-actin was used as a loading control. b Cell viability of SK-N-DZ, IGR-N91 and IGR-NB8 was measured using an MTT assay pre and post HCQ treatment (30 μM). c Confocal microscopy analysis was done on transfected IGR-N91 cells with GFP-LC3 not treated or treated 7 h with HCQ 30 μM, vincristine 1 μM or the combination of the two drugs. d Autophagy in SK-N-DZ, IGR-N91 and IGR-NB8 cells was measuredbyanMDC assaypre andpostHCQ treatment(30 μM). e SK-N-DZ cell viability was measured after vincristine or doxorubicin treatment combined or not with HCQ (30 μM). f The autophagic activity of SK-N-DZ cells was measured using a MDC agent after treatment with vincristine or doxorubicin, combined or not with HCQ (30 μM). Results are expressed as percentage of corresponding control and represent mean ± SEM of 4 independent experiments (*: P < 0.05, **: P < 0.01, ***: P < 0.001) pAKT, suggesting that autophagy activation occurs after increased autophagy comparing to other cell lines with inhibition of the AKT pathway since the inhibition of non-amplified MYCN. Temozolomide has been de- mTOR activates autophagy. scribed as an inducer of autophagy [35]. In the present To determine whether autophagy is activated after study, temozolomide doesn’t induce a dose-dependent chemotherapy treatment, three chemotherapeutic increase of autophagy, detectable by MDC and by agents, used clinically for NB treatment, were studied: Western blot. Differently, the present study showed that vincristine, doxorubicin and cisplatin. Our data demon- LY294002 was a dose dependent activator of autophagy. strate that the activation of autophagy by cisplatin and This drug increases autophagy by inhibiting PI3K I, an vincristine is dose-dependent. This is also observed with element of the PI3K/AKT/mTOR inhibitory pathway to doxorubicin except in one cell line (SK-N-FI). These autophagy [36]. Rapamycin induces autophagy by data were confirmed by in vivo study that show the acti- inhibiting mTOR [37]. In the present study, an increase vation of autophagy in NB tumors from mice receiving in autophagy detected by MDC was associated with the cisplatin or vincristine treatment compared to non- treatment by rapamycin in the four studied NB cell lines. treated tumors. In other cancers, doxorubicin is known It has been described that in the presence of rapamycin, as an inducer of the autophagosome formation in the dephosphorylation of many proteins activates the papillary thyroid cancer [32], whereas cisplatin induced transcription of ATG8 and ATG14 genes, known to be autophagy in esophageal squamous cell carcinoma cells associated with autophagy [13]. [33]. The MYCN gene amplification in several cell lines Beclin 1 has many cell functions, such as inducing used in our study could change the state of autophagy autophagosome formation and acting as a tumor sup- indeed several studies have showed that overexpression pressor [38]. It is a mediator of various cellular cascades, of CMYC strongly induces autophagy in rat 3Y1 fibro- including autophagy, apoptosis and cell differentiation blasts [33, 34]. Our data indicate that NB cell lines with [39]. The association of certain genes of the Bcl-2 family amplified MYCN (SK-N-DZ, IGR-N91) didn’t show with Beclin 1 inhibits the autophagic function by Fig. 5 Effect of autophagy inhibition by HCQ on tumors in vivo. Six NSG mice of each group received a s.c injection of 5x10 IGR-N91 cells. When tumors reached 100 mm , mice were administered vincristine (0.4 mg/kg/day), HCQ (60 mg/kg), an association of vincristine and HCQ or vehicle (saline) for 9 days. Thetumor size of thefourgroupsofmicewas compared during this period Belounis et al. BMC Cancer (2016) 16:891 Page 12 of 14 limiting its partnering with a class III PI3K and activates HCQ sensitizes cells to the conventional chemotherapy its apoptotic function instead [40]. It is the positive in NB treatment. Therefore, we propose that HCQ could regulation of Beclin 1 that induces the autophagic and be used as an adjuvant therapy in clinical trials to differentiation cascades [39]. Also, Beclin 1 loses its au- enhance the efficacy of chemotherapy in NB patients. tophagic abilities and induces apoptosis when there is an increased activity of caspase-9 [41]. This caspase has the Additional files property of cleaving Beclin 1 and creating a C-terminal fragment promoting pro-apoptotic activity [41]. Accord- Additional file 1: Figure S1. Control for immunogistochemistry. Normal mouse or rabbit IgG at the same concentration as the primary ing to some studies, doxorubicin activates caspase-9 and antibody were used as negative control (NC) and synaptophysin (1/100, positively regulates the expression of Beclin 1 [41]. Con- Polyclonal, SP11, Thermofisher Scientific) as positive control (PC). sequently, this particular NB drug induces apoptosis. (PDF 379 kb) Collectively, our data demonstrate that autophagy is acti- Additional file 2: Figure S2. GFP-LC3 transfection and confocal microscopy. The cell line IGR-N91 transfected with GFP-LC3 were not vated in response to chemotherapy in NB cells but has not treated (a), treated with vincristine alone (1 μM) (b), with HCQ (30 μM) answered if this autophagy is an autophagy-mediated cell alone (c) or with the association of the two drugs (d) and then analyzed death or autophagy-mediated cell survival (chemoresis- with confocal microscopy for 7 h. Treatment with HCQ induced an inhibition of the late stage of autophagy which was shown an tance). We investigated the effect of combining chemo- accumulation of autophagosome. (PDF 789 kb) therapy with autophagy inhibition. In the early stages of Additional file 3: Figure S3. HCQ sensitizes NB cells to chemotherapy carcinogenesis, autophagy acts as a primary tumor suppres- by inhibition of autophagy. A. N91-IGR or NB8-IGR cell viability was sor and inhibits tumor progression [21]. However, once measured after vincristine or doxorubicin treatment combined or not to HCQ 30 µM. Results are expressed as percentage of corresponding control tumor is established, autophagy tend to be a protective and represent mean ± SEM of 4 independent experiments. B. Autophagic mechanism used by tumors cells to overcome chemother- activity of N91-IGR or NB8-IGR was measured using MDC agent after apy and other metabolic stress. We used HCQ to target the increasing concentration of vincristine or doxorubicin treatment combined or not to HCQ 30 µM. Fluorescence was quantified by spectrophotometer. late stages of autophagy by blocking the fusion of autopha- (*: P <0.05), (**: P <0.01), (***: P < 0.001). (PDF 215 kb) gosomes with lysosomes [42]. Interestingly, mice treated Additional file 4: Figure S4. Effect of ATG5 knockdown xenografts on with HCQ alone have decreased tumors volume compared tumor growth. (11/12) NSG Mice developed xenograft tumor from s.c to mice treated with vincristine. This results support some injection of 5.10 sheGFP cells and only (3/12) from shATG5 cells (Table A). When tumor reached 200 mm3, mice were administered vincristine clinical trials that use HCQ alone to treat patients with (0.4 mg/kg/day) or vehicle (saline) for 5 days. Progression of tumor pancreatic cancer, breast cancer, renal cell carcinoma or volume was followed in each group. Each data point represents the chronic lymphocytic leukemia [23, 43]. In the present study, mean 6 ± SE tumor volume for sheGFP cells and 1 or 2 for shAtg5 cells. At day 6, Tumors size from shATG5 cells were reduced compared with HCQ was used at non-toxic concentrations that trigger tumors from eGFP (B). Also, tumors from shATG5 cells were more autophagy inhibition. Our data demonstrated that mice sensitive to vincristine comparing to control cells. Immunohistochemistry treated with a combination of vincristine and HCQ develop was performed on the sections of tumors developed in the mouse model. with LC3 antibody and showed a high expression of LC3 II in tumors with a significantly less extend than mice treated control cells and a very low in cells ATG5 knockdown as well as treated with vincristine alone. These data demonstrate, for the first (b, d) or not treated (a, c) (C). (PDF 259 kb) time, that inhibition of autophagy using HCQ sensitizes NB cells to classical chemotherapy supporting the idea that au- Abbreviations tophagy acts as a cytoprotective mechanism [44] and its in- ATG: Autophagy-related genes; Cis: Cisplatin; CQ: Chloroquine; DXR: Doxurubicin; HCQ: Hydroxycholoroquine; IHC: Immunohistochemistry; hibition may promote apoptosis in cancer cells [45]. Several LC3: Microtubule-associated protein light chain 3; studies and clinical trials have investigated autophagy MDC: Monodansylcadaverine; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5- inhibition using different pharmacological agents, usually in diphenyltetrazolium bromide; NB: Neuroblastoma; NSG: NOD scid gamma; shRNA: Short-hairpin RNA; TMA: Tissue microarray; VCR: Vincristine combination with chemotherapy, radiotherapy or other tar- geted anti-cancer therapies [46]. Some data demonstrated a Acknowledgements significant increase in long-term survival when the treat- This work was supported by grants from the Fondation Centre de ment includes CQ [47]. Reports from clinical trials indicate Cancérologie Charles-Bruneau, Canada and the Comité de Montbéliard ligue contre le cancer, France. We also thanks Dr Christian Beauséjour for providing that when CQ was added to conventional therapy, im- packaging plasmids. provement of mid-term survival for glioblastoma multiform patients was seen [48]. Taken together, these results Funding This study was supported by Fondation Centre de Cancérologie Charles-Bruneau, confirm that autophagy promotes chemoresistance and its Montreal, Canada and the Comité de Montbéliard ligue contre le cancer, France. inhibition sensitizes NB cells to chemotherapy. Availability of data and materials Conclusions All chemicals, reagents, drugs and plasmids were purchased. No chemicals or plasmids were formulated or manufactured in our laboratory, Our study demonstrates that autophagy is present in NB All data and results are already present in the manuscript and supporting tumors at a basal level and it is activated after chemo- files. There is thus no need to provide links to study data. The cell lines therapeutic that confers chemoresistance. The use of described in this study are available to investigators upon request. Belounis et al. BMC Cancer (2016) 16:891 Page 13 of 14 Authors’ contributions 15. Galluzzi L, et al. Guidelines for the use and interpretation of assays for monitoring All authors have read and approved the final text of the manuscript. AB cell death in higher eukaryotes. Cell Death Differ. 2009;16(8):1093–107. carried out the majority of the in vitro study (MTT, Immunoblot and 16. Miracco C, et al. Beclin 1 and LC3 autophagic gene expression in cutaneous autophagy assay), interpretation of the data, statistical analysis, and melanocytic lesions. Hum Pathol. 2010;41(4):503–12. manuscript drafting. CN conceived of the study, and participated in its 17. Fujiwara K, et al. Akt inhibitor shows anticancer and radiosensitizing effects in design and coordination and helped to draft the manuscript. RLG carried out malignant glioma cells by inducing autophagy. Int J Oncol. 2007;31(4):753–60. the in vivo study. MM carried out the production of lentiviral particles and 18. Petiot A, et al. Distinct classes of phosphatidylinositol 3'-kinases are involved transduction of NB cells. PT involved in drafting the manuscript or revising it in signaling pathways that control macroautophagy in HT-29 cells. J Biol critically for important intellectual content. MB and SC participated in the in Chem. 2000;275(2):992–8. vivo study. ÉH and GV contributed in results interpretation. HS carried out 19. Budanov AV, Karin M. p53 target genes sestrin1 and sestrin2 connect the interpretation of TMA, conceived of the study and participated in its genotoxic stress and mTOR signaling. Cell. 2008;134(3):451–60. design and coordination and helped to draft the manuscript. All authors 20. Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged read and approved the final manuscript. sword. Science. 2004;306(5698):990–5. 21. Turcotte S, Giaccia AJ. Targeting cancer cells through autophagy for Competing interests anticancer therapy. Curr Opin Cell Biol. 2010;22(2):246–51. The authors declare that they have no competing interests. 22. Paglin S, et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 2001;61(2):439–44. 23. Yang ZJ, et al. The role of autophagy in cancer: therapeutic implications. Consent for publication Mol Cancer Ther. 2011;10(9):1533–41. Not applicable. 24. Brodeur GM, et al. International criteria for diagnosis, staging, and response to treatment in patients with neuroblastoma. J Clin Oncol. 1988;6(12):1874–81. Ethics approval and consent to participate 25. Biederbick A, Kern HF, Elsasser HP. Monodansylcadaverine (MDC) is a specific in Written informed consent for the use of tissues for research was obtained vivo marker for autophagic vacuoles. Eur J Cell Biol. 1995;66(1):3–14. from patient guardians. The study was reviewed and approved by the 26. Burkhart CA, et al. Small-molecule multidrug resistance-associated protein 1 Research Ethics Board of the Sainte-Justine University Health Center. inhibitor reversan increases the therapeutic index of chemotherapy in This study employed animals and thus the general guidelines established by mouse models of neuroblastoma. Cancer Res. 2009;69(16):6573–80. the European Community (Directive 2010/63/UE) were followed, while the 27. Harned TM, et al. Sodium thiosulfate administered six hours after cisplatin experimental protocols were approved by the Institutional Committee for does not compromise antineuroblastoma activity. Clin Cancer Res. Ethics in Animal Research (CEEA26. Approval number: 2013–099). 2008;14(2):533–40. 28. Das B, et al. Squalene selectively protects mouse bone marrow progenitors Author details against cisplatin and carboplatin-induced cytotoxicity in vivo without Research centre of the Sainte Justine university hospital, Montreal, QC, protecting tumor growth. Neoplasia. 2008;10(10):1105–19. Canada. Department of pathology and cellular biology, Université de 29. Sui X, et al. Autophagy and chemotherapy resistance: a promising Montréal, Montreal, QC, Canada. Department of biochemistry, CHU Sainte therapeutic target for cancer treatment. Cell Death Dis. 2013;4:e838. Justine, Montreal, QC, Canada. Division of paediatric oncology, CHU Sainte 30. Wang J, et al. Aberrant expression of Beclin-1 and LC3 correlates with poor Justine, Montreal, QC, Canada. Department of surgery, CHU Sainte Justine, prognosis of human hypopharyngeal squamous cell carcinoma. PLoS One. 3175 Montreal, QC, Canada. Department of paediatric oncology, Institut 2013;8(7):e69038. Gustave Roussy, Villejuif, France. Department of pathology and cytogenetic, 31. Ma XH, et al. Measurements of tumor cell autophagy predict invasiveness, CHU Sainte Justine, Montreal, QC, Canada. resistance to chemotherapy, and survival in melanoma. Clin Cancer Res. 2011;17(10):3478–89. Received: 4 April 2016 Accepted: 27 October 2016 32. Lin CI, et al. Autophagy: a new target for advanced papillary thyroid cancer therapy. Surgery. 2009;146(6):1208–14. 33. Liu D, et al. Inhibition of autophagy by 3-MA potentiates cisplatin-induced References apoptosis in esophageal squamous cell carcinoma cells. Med Oncol. 1. Schwab M, et al. Neuroblastoma: biology and molecular and chromosomal 2011;28(1):105–11. pathology. Lancet Oncol. 2003;4(8):472–80. 34. Tsuneoka M, et al. c-myc induces autophagy in rat 3Y1 fibroblast cells. Cell 2. Canadian Cancer Society's Steering Commitee. Canadian Cancer Statistics Struct Funct. 2003;28(3):195–204. 2009. 2009. 35. Kanzawa T, et al. Role of autophagy in temozolomide-induced cytotoxicity 3. Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat for malignant glioma cells. Cell Death Differ. 2004;11(4):448–57. Rev Cancer. 2003;3(3):203–16. 36. Xing C, et al. Class I phosphatidylinositol 3-kinase inhibitor LY294002 activates 4. Maris JM. The biologic basis for neuroblastoma heterogeneity and risk autophagy and induces apoptosis through p53 pathway in gastric cancer cell stratification. Curr Opin Pediatr. 2005;17(1):7–13. line SGC7901. Acta Biochim Biophys Sin (Shanghai). 2008;40(3):194–201. 5. Park JR, Eggert A, Caron H. Neuroblastoma: biology, prognosis, and 37. Chang YY, et al. Nutrient-dependent regulation of autophagy through the treatment. Pediatr Clin North Am. 2008;55(1):97–120. x. target of rapamycin pathway. Biochem Soc Trans. 2009;37(Pt 1):232–6. 6. Matthay KK, et al. Treatment of high-risk neuroblastoma with intensive 38. Pirtoli L, et al. The prognostic role of Beclin 1 protein expression in high- chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13- grade gliomas. Autophagy. 2009;5(7):930–6. cis-retinoic acid. Children's Cancer Group. N Engl J Med. 1999;341(16):1165–73. 39. Wang J. Beclin 1 bridges autophagy, apoptosis and differentiation. 7. Maris JM, et al. Neuroblastoma. Lancet. 2007;369(9579):2106–20. Autophagy. 2008;4(7):947–8. 8. He C, Klionsky DJ. Regulation mechanisms and signaling pathways of 40. Wei Y, et al. JNK1-mediated phosphorylation of Bcl-2 regulates starvation- autophagy. Annu Rev Genet. 2009;43:67–93. induced autophagy. Mol Cell. 2008;30(6):678–88. 9. Klionsky DJ. Autophagy: from phenomenology to molecular understanding 41. Furuya D, et al. Beclin 1 augmented cis-diamminedichloroplatinum induced in less than a decade. Nat Rev Mol Cell Biol. 2007;8(11):931–7. apoptosis via enhancing caspase-9 activity. Exp Cell Res. 2005;307(1):26–40. 10. Reggiori F, Klionsky DJ. Autophagosomes: biogenesis from scratch? Curr 42. Ben-Zvi I, et al. Hydroxychloroquine: from malaria to autoimmunity. Clin Rev Opin Cell Biol. 2005;17(4):415–22. Allergy Immunol. 2012;42(2):145–53. 11. Chen D, et al. A mammalian autophagosome maturation mechanism mediated by TECPR1 and the Atg12-Atg5 conjugate. Mol Cell. 2012;45(5):629–41. 43. Lagneaux L, et al. Early induction of apoptosis in B-chronic lymphocytic 12. Eskelinen EL, Saftig P. Autophagy: a lysosomal degradation pathway with a leukaemia cells by hydroxychloroquine: activation of caspase-3 and no central role in health and disease. Biochim Biophys Acta. 2009;1793(4):664–73. protection by survival factors. Br J Haematol. 2001;112(2):344–52. 13. Yang YP, et al. Molecular mechanism and regulation of autophagy. Acta 44. Shen S, et al. Association and dissociation of autophagy, apoptosis and Pharmacol Sin. 2005;26(12):1421–34. necrosis by systematic chemical study. Oncogene. 2011;30(45):4544–56. 14. Yu L, Strandberg L, Lenardo MJ. The selectivity of autophagy and its role in 45. Maiuri MC, et al. Self-eating and self-killing: crosstalk between autophagy cell death and survival. Autophagy. 2008;4(5):567–73. and apoptosis. Nat Rev Mol Cell Biol. 2007;8(9):741–52. Belounis et al. BMC Cancer (2016) 16:891 Page 14 of 14 46. White E, DiPaola RS. The double-edged sword of autophagy modulation in cancer. Clin Cancer Res. 2009;15(17):5308–16. 47. Liang X, et al. Inhibiting systemic autophagy during interleukin 2 immunotherapy promotes long-term tumor regression. Cancer Res. 2012;72(11):2791–801. 48. Sotelo J, Briceno E, Lopez-Gonzalez MA. Adding chloroquine to conventional treatment for glioblastoma multiforme: a randomized, double- blind, placebo-controlled trial. Ann Intern Med. 2006;144(5):337–43. 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Published: Nov 15, 2016

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