Proteomic signature of neuroblastoma cells UKF-NB-4 reveals key

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Proteomic signature of neuroblastoma cells UKF-NB-4 reveals key role of lysosomal sequestration and the proteasome complex in acquiring chemoresistance to cisplatin Miguel Angel Merlos Rodrigo, Hana Buchtelova, Vivian de los Rios, Jose Ignacio Casal, Tomas Eckschlager, Jan Hrabeta, Marie Belhajova, Zbynek Heger, and Vojtech Adam J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00867 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 7, 2019

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Journal of Proteome Research

Proteomic signature of neuroblastoma cells UKF-NB-4 reveals key role of lysosomal sequestration and the proteasome complex in acquiring chemoresistance to cisplatin

Miguel Angel Merlos Rodrigo1,2, Hana Buchtelova1,2, Vivian de los Rios3, José Ignacio Casal3, Tomas Eckschlager4, Jan Hrabeta4, Marie Belhajova4, Zbynek Heger1,2, Vojtech Adam1,2*

1Department

of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00

Brno, Czech Republic 2Central

European Institute of Technology, Brno University of Technology, Purkynova 123,

612 00 Brno, Czech Republic 3Functional Proteomics, Department of Molecular Biomedicine and Proteomic Facility, Centro

de Investigaciones Biológicas (CIB-CSIC), Ramiro de Maeztu 9, Madrid 280 40, Spain 4Department

of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles

University, and University Hospital Motol, V Uvalu 84, 150 06 Prague 5, Czech Republic

*Corresponding author Vojtech Adam, Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic; E-mail: [email protected]; phone: +420-5-4513-3350; fax: +420-5-4521-2044.

1 ACS Paragon Plus Environment

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Abstract Cisplatin (CDDP) is a widely used agent in the treatment of neuroblastoma. Unfortunately, the development of acquired chemoresistance limits its clinical use. To gain a detailed understanding of the mechanisms underlying the development of such chemoresistance, we comparatively analysed established cisplatin-resistant neuroblastoma cell line (UKF-NB4CDDP) and its sensitive counterpart (UKF-NB-4). First, using viability screenings, we confirmed the decreased sensitivity of tested cells to cisplatin and identified a cross-resistance to carboplatin and oxaliplatin. Then, the proteomic signatures were analysed using nanoLC MS/MS. Among the proteins responsible for UKF-NB-4CDDP chemoresistance, ion channels transport family proteins, ABC superfamily proteins, SLC-mediated trans-membrane transporters, proteasome complex subunits and V-ATPases were identified. Moreover, we detected markedly higher proteasome activity in UKF-NB-4CDDP cells and a remarkable lysosomal enrichment that can be inhibited by bafilomycin A to sensitize UKF-NB-4CDDP to CDDP. Our results indicate that lysosomal sequestration and proteasome activity may be one of key mechanisms responsible for intrinsic chemoresistance of neuroblastoma to CDDP.

Keywords: Neuroblastoma; Chemoresistance; Cisplatin; Lysosomes; Proteomics; V-ATPases


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Introduction Neuroblastoma (Nbl) is an embryonal tumour, originating from progenitor cells of the sympathetic nervous system, and is the most common extra-cranial solid tumour of childhood 1-3.

Nbl accounts for 7% of malignancies from birth to 14-y of age 4 and 12% of cancer deaths

in children. Biology of Nbl is heterogeneous, only a minimal number of Nbl regress spontaneously, while in the majority of the cases, the development of a malignant phenotype characterized by aggressive behaviour with metastasis results in mortality of more than 50% of the chemoradiotherapy-treated patients 5-8. Current treatment strategies for high-risk neuroblastoma (H-R Nbl) involve intensive cytotoxic induction chemotherapy followed by surgical resection of gross disease and/or radiotherapy, megachemotherapy followed with autologous hematopoietic progenitor cells transplantation and maintenance therapy

9.

Cisplatin [cis-diamminedichloroplatinum (CDDP)] and/or

carboplatin belong among the most common agents used in Nbl therapy

10-14.

Unfortunately,

due to often acquired cisplatin-chemoresistance, the prognosis of H-R Nbl patients after CDDP treatment remains poor. CDDP acts on several signalling pathways, triggering mechanisms that are involved in resistance development by establishing complicated self-defence system(s) to escape exogenous cytotoxic compounds of different origins 15. Among these systems, multidrug resistant transporters, defective endocytic uptake, aberrant promoter hypermethylation and histone modifications, epithelial to mesenchymal transition or a plethora of miRNAs have been identified in distinct types of cancers (summarized in 16). Several high-throughput studies considerably contributed to collect large amount of data on possible mechanisms underlying CDDP resistance in different cell models

5, 17-19.

Our pilot

transcriptomic study revealed that the transient up-regulation of human metallothionein-3 (hMT-3) in wild-type Nbl cells (SiMa) elicits chemoresistance to CDDP through dysregulating genes involved in oncogene-induced senescence and apoptosis 19. D’Aguanno and co-workers



identified that the regulation of sensitivity to CDDP in SH-SY5Y cells may be driven by Nrf2 pathway 5. Similarly, Tabata et al. identified that acute application of CDDP can alter expression of enzymes involved in DNA methylation status 18. However, due to the limitations of 2D-electrophoresis, it can be expected that the low abundance, very large or small proteins cannot be properly identified. Therefore, to unravel the putative mechanisms responsible for acquired chemoresistance to CDDP in UKF-NB-4 cells, we carried out comparative deep proteome survey using nanoLC MS/MS. The obtained results indicate that due to the induced chemoresistance, UKF-NB-4CDDP cells acquire a highly specific proteomic signature that promotes a remarkable lysosomal enrichment together with the increased activity of proteasome complex.

Materials and Methods Chemicals All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) in ACS purity, unless noted otherwise.

Cell lines and culture conditions The parental UKF-NB-4 cell line, established from recurrent bone marrow metastases of H-R Nbl and the cisplatin-resistant line UKF-NB-4CDDP were a kind gift from Prof. J. Cinatl (Goethe University in Frankfurt am Main, Germany). The cells were cultured in IMDM supplemented with 10% foetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA). The cell cultures were incubated at 37°C in a humidified 5% CO2 atmosphere. The cell lines were passaged at regular intervals twice a week. The morphology of cells was observed under phase contrast using EVOS FL Auto 2 (Thermo Fisher Scientific).


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Alamar blue cytotoxicity assay For dose-response curves, cells were seeded in 100 µL of IMDM at a density of 104 cells per well. To investigate the effect of CDDP, carboplatin and oxaliplatin, the cells were treated either with 0.29-1000 µM of CDDP, oxaliplatin and/or carboplatin. To evaluate the synergistic effects, cytotoxicity of CDDP (20 µM) and bafilomycin A (0.1 µM) and their co-treatments were studied, too. The viability was evaluated by AlamarBlue® test as previously described by Ahmed et al.

20.

Briefly, after 48 h treatment 10 µL AlamarBlue® Cell Viability reagent

(Thermo Fisher Scientific) was added and the plates were incubated at 37°C for 2 h. The fluorescence (excitation wavelength 565 nm, emission wavelength 610 nm) was analysed for each well by multiwell SpectraMax® i3x Platform (Molecular Devices, Sunnyvale, CA, USA). The values of 48IC50 were determined utilizing the linear regression of the dose-log response curves by SOFTmaxPro software.

Sample preparation and nLC-MS/MS analysis After protein extraction using RIPA buffer, proteins were reduced, alkylated, and digested with trypsin overnight at 37°C. Resulting peptides were desalted and separated on an Easy-nLC 1000 nano system (Thermo Scientific). Then, the samples were loaded into a precolumn Acclaim PepMap 100 (Thermo Scientific) and eluted in a RSLC PepMap C18, 25 cm long, 75 µm inner diameter and 2 µm particle size (Thermo Scientific). The mobile phase flow rate was 300 nL/min using 0.1% formic acid in water (v/v) and 0.1% formic acid (v/v) and 100% acetonitrile (v/v). MS analysis was performed using a Q-Exactive mass spectrometer (Thermo Scientific). For ionization, 2000 V of liquid junction voltage and 270°C capillary temperature was used. The full scan method employed a m/z 400–1500 mass selection, an Orbitrap resolution of 70.000 (at m/z 200), a target automatic gain control (AGC) value of 3e6, and maximum injection times of 100 ms. After the survey scan, the 15 most intense precursor ions were selected for



MS/MS fragmentation. Fragmentation was performed with a normalized collision energy of 27 eV and MS/MS scans were acquired with a starting mass of m/z 100, AGC target was 2e5, resolution of 17,500 (at m/z 200), intensity threshold of 8e3, isolation window of 2 m/z units. Charge state screening was enabled to reject unassigned, singly charged, and equal or more than seven protonated ions. A dynamic exclusion time of 20 s was used to discriminate against previously selected ions. The protein identification was carried out by nLC-MS/MS.

MS data analysis MS data were analysed with Proteome Discoverer (v.1.4.1.14) (Thermo Fisher Scientific) using standardized workflows. The mass spectrum *.raw file was searched against the human SwissProt 57.15 database (20,266 sequences protein entries) using MASCOT search engine (version 2.3, Matrix Science). Precursor and fragment mass tolerance were set to 10 ppm and 0.02 Da, respectively, allowing 2 missed cleavages, carbamidomethylation of cysteines as a fixed modification, methionine oxidation, phosphorylation serine, threonine and tyrosine acetylation N-terminal and as a variable modification. Identified peptides were filtered using Percolator algorithm 21, 22 with a q-value threshold of 0.01.

Western Blotting The cells were harvested by trypsination and centrifuged at 10,000 rpm for 10 min. The lysis was done on ice with 200 µL of RIPA lysis buffer containing 10 µL of protease inhibitor cocktail. Equal amounts of protein were separated using SDS-PAGE and then electroblotted onto polyvinylidene fluoride membrane. The membrane was blocked with 1% dried skimmed milk in phosphate buffered saline pH 7.4 and then incubated separately at 4°C with indicated primary antibodies (all Abcam, Cambridge, UK, dilution 1:750) overnight. Further, the membrane was incubated with peroxidase-conjugated secondary antibodies (1:1000) for 1 h at


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25°C. The bands were developed using LuminataTM Forte substrate (EMD Millipore, Burlington, MA, USA). Blots were visualized using Azure c600 (Azure Biosystems, Dublin, CA, USA).

Proteasome complex activity assay Proteasome complex activity was analysed by Proteasome Activity Assay Kit (Abcam). After the 48 h treatment with CDDP (relevant 48IC50), fluorescence (λexc 350 nm, λem 440 nm) of cells was analysed using a SpectraMax® i3x Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) in the presence or absence of specific proteasome inhibitor MG132 after 5 min at 37°C for 30 min.

LysoTracker® red (LTR) uptake The day before analysis, the cells were seeded at 1 × 104 cells per well in 96-well plate. Cells were treated with 200 nM LTR (Thermo Fisher Scientific) for 30 min at 37°C and fluorescence intensity of LTR in individual cells (object average intensity) was examined (λexc 541 nm, λem 714/108 nm) with a SpectraMax® i3x Multi-Mode Microplate Reader (Molecular Devices) using imaging cytometer module. Twelve samples were analysed in parallel from each line, and three independent experiments were performed.

Confocal laser scanning microscopy (CLSM) The cells were grown on 35 mm glass bottom culture dishes (In Vitro Scientific, Sunnyvale, CA, USA) for 24 h before treatment. The cells were treated with 100 nM LTR (for 30 minutes at 37°C) and nuclei were counterstained with Hoechst 33342. The cells were observed under a CLSM Leica TCS SP8 (Leica Microsystems GmbH, Wetzlar, Germany). For excitation of LTR,



laser with wavelength of 552 nm was used; emitted light was collected in the range of 580–620 nm. All images were recorded with ×100 objective using the Leica Application Suite X system.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) qRT-PCR was performed for human V-ATPase subunits (ATP6V0D1, ATP6V0A1, TP6V0E1, ATP6V0B, ATP6V0C, ATP6V1A1, ATP6V1B2, ATP6V1C1, ATP6V1F, ATP6V1E2, TP6V1D, ATP6V1H and ATP6V1G1) as the target genes and POLR2A as the reference gene. An isolation of total RNA was performed using a PureLink RNA Mini Kit (Thermo Fisher Scientific) according to producer´s protocol. Complementary DNA was synthesized from 1000 ng of RNA by Generi Biotech Reverse Transcription Kit (Generi Biotech Hradec Kralove, Czech Republic). The primers and probes were designed and produced by Generi Biotech. Each reaction (20 μL) consisted of 10 μL of universal master mix, 2 μL of primer with fluorescein label, 7 μL of RNAse free water (all Generi Biotech) and 1 μL of cDNA sample. Samples were analysed the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) in triplicate using following conditions: 95°C for 3 min, 50 cycles with the following parameters: 95°C for 10 s, 60°C for 20 s. Relative expression was determined using REST 2009 software. Analyses were performed three times of the three independent samples.

Descriptive statistics and exploited bioinformatic tools For the statistical evaluation of the results, the mean was taken as the measurement of the main tendency, while standard deviation was taken as the dispersion measurement. Differences between groups were analysed using paired t-test and ANOVA. Unless noted otherwise, the threshold for significance was p < 0.05. For analyses Software Statistica 12 (StatSoft, Tulsa, OK, USA) was employed. Expression-based heat maps were constructed using Heatmapper (http://www.heatmapper.ca). The Nbl metabolic pathway was analysed using David Software


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(https://david.ncifcrf.gov/home.jsp) and Panther Classification System (http://pantherdb.org/), which provides golden standard sets of cells component and molecular pathways. The interactome networks were constructed using the STRING software (http://string-db.org/) and the involvement of genes involved in a cellular process was analysed using the Reactome (www.reactome.org).

Results UKF-NB-4CDDP demonstrate a cross-resistance to oxaliplatin and carboplatin In order to verify the acquired chemoresistance to CDDP and cross-resistance to other platinum cytostatics, we investigated the susceptibility of Nbl cells to various platinum coordination compounds. Indeed, we proved a decreased sensitivity of UKF-NB-4CDDP to CDDP and, moreover, we found a developed cross-resistance to carboplatin and oxaliplatin (Fig. 1A). 48IC50 for CDDP and carboplatin was approximately three-fold, and for oxaliplatin 3.8 foldhigher for UKF-NB-4CDDP than for parental cell line (Supplementary File S1). Moreover, we found that UKF-NB-4CDDP cells tend to develop an atypical clumping morphology not observed for UKF-NB-4 (Fig. 1B).

UKF-NB-4CDDP exhibits significantly different proteomic signature In order to investigate the differences in protein expression resulting from CDDP resistance, the deep proteomic signatures were analysed. Out of a total 1802 identified proteins, 1448 were commonly expressed in both tested cell lines. 281 exclusive proteins were registered in the UKF-NB-4CDDP and 73 proteins were exclusively identified in parental UKF-NB-4 cells (Fig. 2A). A full list of proteins as well as relevant MS data are available in Supplementary File S2. Out of total 1448 commonly expressed proteins, 254 were up-regulated (fold ratio >2.5) and 83 were down-regulated (fold ratio

Proteomic signature of neuroblastoma cells UKF-NB-4 reveals key

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