proteomic analysis of the non-genetic response to

Abstract. Background: Drug resistance is the main cause oftherapy failure in advanced lung cancer. Although non-geneticmechanisms play important roles in tumor chemoresistance,drug-induced epigenetic reprogramming is still poorlyunderstood. Materials and Methods: The A549 cell line wasused to generate cells with non-genetic resistance to cisplatin(CDDP), namely A549/CDDP cells. Bioorthogonal non-canonical amino acid tagging (BONCAT) and massspectrometry were used to identify proteins modulated byCDDP in A549 and A549/CDDP cells. Results: Proteinsrelated to proteostasis, telomere maintenance, cell adhesion,cytoskeletal remodeling, and cell redox homeostasis werefound enriched in both cell lines upon CDDP exposure. Onthe other hand, proteins involved in drug response, metabolicpathways and mRNA processing and splicing were up-regulated by CDDP only in A549/CDDP cells. Conclusion:Our study revealed proteome dynamics involved in the non-genetic response to CDDP, pointing out potential targets tomonitor and overcome epigenetic resistance in lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause ofcancer-related death in the world (1). Cisplatin (CDDP)-based chemotherapy is the standard first-line treatment for

inoperable, advanced NSCLC (2). However, drug resistanceremains a major challenge for successful treatment. Cellmodels have been widely used to study cancer drugresistance (3, 4). However, the cell models with high levelsof resistance used in most studies have shown limitedtranslation to clinical application (5). In fact, cell linesconsidered clinically relevant exhibit resistance levels similarto those found in cells isolated from patients afterchemotherapy (from 2- to 5-fold increase in resistance) (3).These clinically relevant models often show unstableresistance, which suggests that resistance acquisition ismainly due to gene expression reprogramming instead ofgenetic alterations.

Accumulating evidence has shown that non-geneticmechanisms play an important role in the chemoresistanceof a variety of tumors (6, 7). During non-genetic evolution,gene expression programs that improve cancer celladaptability and survival are selected and/or induced by drugtreatment (7-9). However, drug-induced epigeneticreprogramming is still poorly characterized, mainly becauseit is a highly dynamic process whose analysis is technicallychallenging.

Bioorthogonal non-canonical amino acid tagging(BONCAT) is a powerful tool to monitor protein dynamicsin response to a wide variety of stimuli. In the BONCATmethod, an artificial amino acid (e.g. L-azidohomoalanine, asurrogate for L-methionine) carrying an azide or alkynegroup is incorporated into newly synthesized proteins, thusallowing for selective detection or purification of taggedproteins by click chemistry (10, 11).

Herein, we performed a proteomic analysis of non-geneticresistance to CDDP in lung cancer cells. We used BONCAT andliquid chromatography-tandem mass spectrometry (LC-MS/MS)in a time-course analysis to selectively label, purify and identifyproteins synthesized by CDDP-sensitive and -resistant A549cells in response to drug exposure.

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This article is freely accessible online.

Correspondence to: Karina Mariante Monteiro, Centro deBiotecnologia, Universidade Federal do Rio Grande do Sul, CaixaPostal 15005, 91501-970 Porto Alegre, RS, Brazil. E-mail:[email protected]

Key Words: BONCAT, cisplatin, drug resistance, lung cancer,proteomics.

©2021 International Institute of Anticancer Research www.iiar-anticancer.org

Proteomic Analysis of the Non-genetic Response to Cisplatin in Lung Cancer CellsCRISTINE DE SOUZA DUTRA1, CAROLINA LUMERTZ MARTELLO1, NATHAN ARAUJO CADORE1, HENRIQUE BUNSELMEYER FERREIRA1,2, ARNALDO ZAHA1,2 and KARINA MARIANTE MONTEIRO1,2

1Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil;2Departamento de Biologia Molecular e Biotecnologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

CANCER DIAGNOSIS & PROGNOSIS1: 235-243 (2021) doi: 10.21873/cdp.10032

Materials and MethodsCell culture and chemoresistance induction. An in vitro cellularmodel for studying non-genetic resistance to CDDP was developedfrom A549 cells. A549 cells were maintained in RPMI 1640medium supplemented with 10% fetal bovine serum (FBS), 100units/ml penicillin and 100 μg/ml streptomycin in a humidifiedatmosphere of 5% CO2 at 37˚C. The CDDP-resistant subline,A549/CDDP, was obtained by a stepwise drug selection protocol, inwhich A549 parental cells (5×105) were continuously exposed toincreasing concentrations of CDDP (0.1, 0.2, 0.3, 0.4 and 0.5 μMin 0.9% NaCl solution) for 72 h each. A549/CDDP cells wereindependently generated three times to produce the biologicalreplicates used in the experiments. CDDP-resistant cells weremaintained in culture medium containing 0.5 μM of CDDP to

maintain the resistant phenotype. CDDP cytotoxicity wasdetermined by sulforhodamine B (SRB) assay (12).

Metabolic labeling and click enrichment of newly synthesizedproteins. For BONCAT assay, A549 and A549/CDDP cells wereseeded at a density of 3×105 cells/well into 6-well plates andcultured for 18 h. Cells were conditioned in methionine-free RPMImedium (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at37˚C to deplete methionine reserves, then the proteins weremetabolically labeled with azidohomoalanine (AHA, 1 mM) for 2,4 or 8 h in the absence or in the presence of CDDP. Each cell linewas exposed to CDDP concentrations corresponding to theirrespective IC50 values. Cells were lysed and newly synthesizedproteins were enriched using the Click-iT Protein Enrichment Kit(Thermo Fisher Scientific), as described previously (11). Briefly,

CANCER DIAGNOSIS & PROGNOSIS 1: 235-243 (2021)

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Figure 1. Development of non-genetic resistance to CDDP in A549 cells. (A) Dose-response curve and calculated IC50 values. Values represent themean±SD from biological triplicates. (B) Biological processes found enriched in the steady-state proteome of A549 and A549/CDDP cells. The dotcolor indicates the significance of the enrichment [-log10 (FDR- corrected p-values)] and the dot size indicates the number of proteins associatedwith each process. The vertical grey dashed line represents a fold enrichment of 1.

AHA-containing proteins were captured onto an alkyne-agaroseresin and non-specifically bound proteins were removed by washingwith increasingly stringent buffers containing SDS, urea,isopropanol and acetonitrile. The resin-bound proteins were digestedwith trypsin and the generated peptides were desalted using OasisHLB cartridges (Waters Corporation, Milford, MA, USA) followingmanufacturer’s instructions.

Mass spectrometry analysis. Peptides were analyzed by LC-MS/MS using a nanoACQUITY Ultra-Performance LiquidChromatography (UPLC) system coupled to a Xevo G2-XS Q-Tofmass spectrometer (Waters Corporation) with a low-flow probe atthe source. Peptides were separated by analytical chromatography

(Acquity UPLC BEH C18, 1.7 μm, 2.1×50 mm, WatersCorporation) at a flow rate of 8 μl/min, using a 7-85%water/acetonitrile 0.1% formic acid linear gradient over 90 min.The MS survey scan was set to 0.5 s and recorded from 50 to2,000 m/z. MS/MS scans were acquired from 50 to 2000 m/z, andscan time was set to 1 s. Data were collected in data-independentMSE mode. The mass spectrometry data were deposited to theProteomeXchange Consortium via the PRIDE (13) partnerrepository with the dataset identifier PXD021779.

Data analysis and functional annotation. LC-MSE data wereprocessed and searched using ProteinLynx Global Server (PLGS3.0.3, Waters Corporation). The searches were conducted against

Dutra et al: Mechanisms of Non-genetic Resistance to Cisplatin in Lung Cancer

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Figure 2. Overview of proteins and GO terms enriched in A549 and A549/CDDP cells after CDDP exposure. (A) Venn diagram of proteins up-regulated by CDDP in each cell. (B) Biological processes found enriched after drug exposure. Detailed legend is the same as in Figure 1B.


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Table I. Proteins up-regulated after CDDP exposure in A549 and A549/CDDP cells.

Protein name Gene name Accession number A549 A549/CDDP

10 kDa heat shock protein_ mitochondrial HSPE1 P61604 x 14-3-3 protein beta/alpha YWHAB P31946 x14-3-3 protein epsilon YWHAE P62258 x 14-3-3 protein gamma YWHAG P61981 x 14-3-3 protein theta YWHAQ P27348 x 6-phosphogluconate dehydrogenase_ decarboxylating PGD P52209 xActin_ cytoplasmic 2 ACTG1 P63261 x xAdenylyl cyclase-associated protein 1 CAP1 Q01518 xADP-ribosylation factor 5 (Fragment) ARF5 C9J1Z8 xADP/ATP translocase 2 SLC25A5 P05141 xAldehyde dehydrogenase_ dimeric NADP-preferring ALDH3A1 A8MYB8 xAlpha-actinin-4 ACTN4 O43707 x Annexin A4 ANXA4 P09525 x ATP synthase subunit alpha_ mitochondrial ATP5A1 P25705 xATP synthase subunit beta (Fragment) ATP5B H0YH81 x ATP synthase subunit beta_ mitochondrial ATP5B P06576 xATP-citrate synthase ACLY P53396 xATP-dependent RNA helicase DDX3X DDX3X O00571 x Calreticulin (Fragment) CALR K7EJB9 x Cathepsin D CTSD A0A1B0GW44 x xClusterin CLU P10909 x Cofilin-1 CFL1 E9PK25 x xCysteine and histidine-rich domain-containing protein 1 CHORDC1 Q9UHD1 xDestrin DSTN F6RFD5 x Destrin DSTN P60981 xElongation factor Tu_ mitochondrial TUFM P49411 x xEndoplasmic reticulum resident protein 44 ERP44 Q9BS26 xEukaryotic translation initiation factor 5A-1 EIF5A I3L504 x Eukaryotic translation initiation factor 6 EIF6 P56537 x Filamin A FLNA Q60FE5 xFilamin-A FLNA P21333 x xFructose-bisphosphate aldolase A ALDOA P04075 x Galectin-1 LGALS1 P09382 x xGlucose-6-phosphate 1-dehydrogenase (Fragment) G6PD E7EUI8 x Glucose-6-phosphate isomerase (Fragment) GPI A0A0A0MTS2 xGlyceraldehyde-3-phosphate dehydrogenase GAPDH P04406 x xGTP-binding nuclear protein Ran RAN B5MDF5 xGTP-binding protein SAR1a SAR1A Q9NR31 x Heat shock 70 kDa protein 1-like (Fragment) HSPA1L Q53FA3 x Heat shock 70 kDa protein 6 HSPA6 P17066 x Heat shock cognate 71 kDa protein HSPA8 P11142 x xHeme oxygenase 1 HMOX1 P09601 xHeterogeneous nuclear ribonucleoprotein A/B HNRNPAB D6R9P3 xHeterogeneous nuclear ribonucleoprotein A1 HNRNPA1 F8W6I7 xHeterogeneous nuclear ribonucleoprotein D0 (Fragment) HNRNPD H0Y8G5 xHeterogeneous nuclear ribonucleoprotein U HNRNPU Q00839 x xHeterogeneous nuclear ribonucleoproteins C1/C2 HNRNPC G3V4C1 x Histone H2B HIST1H2BN U3KQK0 xHistone H2B type 1-B HIST1H2BB P33778 x xKeratin_ type I cytoskeletal 10 KRT10 P13645 x xKeratin_ type I cytoskeletal 14 KRT14 P02533 x Keratin_ type I cytoskeletal 16 KRT16 P08779 x Keratin_ type I cytoskeletal 18 KRT18 F8VZY9 x Keratin_ type I cytoskeletal 18 KRT18 P05783 x Keratin_ type I cytoskeletal 19 KRT19 P08727 x Keratin_ type I cytoskeletal 19 (Fragment) KRT19 C9JM50 x Keratin_ type I cytoskeletal 9 KRT9 P35527 x x

Table I. Continued


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Table I. Continued

Protein name Gene name Accession number A549 A549/CDDP

Keratin_ type II cuticular Hb1 KRT81 A0A087X106 x Keratin_ type II cytoskeletal 1 KRT1 P04264 x xKeratin_ type II cytoskeletal 2 epidermal KRT2 P35908 x xKeratin_ type II cytoskeletal 7 KRT7 P08729 x Keratin_ type II cytoskeletal 8 KRT8 P05787 x xMalate dehydrogenase_ cytoplasmic MDH1 P40925 x Moesin MSN P26038 xMyosin light polypeptide 6 MYL6 G8JLA2 x Myosin-9 MYH9 P35579 x xNAD(P)H dehydrogenase [quinone] 1 NQO1 H3BRK3 x NAD(P)H dehydrogenase [quinone] 1 NQO1 B4DLR8 xNucleolin NCL P19338 xNucleoside diphosphate kinase NME1-NME2 Q32Q12 xNucleoside diphosphate kinase A NME1 P15531 x Peptidyl-prolyl cis-trans isomerase PPIA C9J5S7 xPeptidyl-prolyl cis-trans isomerase A PPIA P62937 x Peroxiredoxin-1 PRDX1 Q06830 x Peroxiredoxin-1 (Fragment) PRDX1 A0A0A0MSI0 x Phosphoglycerate mutase 1 PGAM1 P18669 xPlastin-3 PLS3 P13797 xPoly(rC)-binding protein 1 PCBP1 Q15365 xPoly(rC)-binding protein 2 (Fragment) PCBP2 H3BRU6 x xPoly(U)-binding-splicing factor PUF60 (Fragment) PUF60 A0A0J9YYL3 x Polypyrimidine tract-binding protein 1 PTBP1 A0A0U1RRM4 xPolyubiquitin-C (Fragment) UBC F5H6Q2 x Prostaglandin reductase 1 PTGR1 Q14914 xProteasome subunit alpha type (Fragment) PSMA4 H0YMA1 xProtein disulfide-isomerase A3 PDIA3 P30101 x xProtein disulfide-isomerase A6 PDIA6 Q15084 xProtein S100-A11 S100A11 P31949 x Putative elongation factor 1-alpha-like 3 EEF1A1P5 Q5VTE0 x xPutative Ras-related protein Rab-1C RAB1C Q92928 x Pyruvate kinase PKM PKM P14618 xRab GDP dissociation inhibitor beta GDI2 P50395 x Ras-related protein Rab-1B RAB1B E9PLD0 xRetinal dehydrogenase 1 ALDH1A1 P00352 xRho-related GTP-binding protein RhoC (Fragment) RHOC E9PQH6 x S-adenosylmethionine synthase isoform type-2 MAT2A P31153 x xSeptin-2 SEPT2 Q15019 x Suppression of tumorigenicity 5 protein (Fragment) ST5 E9PLH6 xT-complex protein 1 subunit alpha TCP1 P17987 xT-complex protein 1 subunit delta CCT4 P50991 x T-complex protein 1 subunit epsilon CCT5 E7ENZ3 xT-complex protein 1 subunit gamma CCT3 P49368 x T-complex protein 1 subunit theta CCT8 P50990 xThioredoxin TXN P10599 x Thymosin beta-4 TMSB4X P62328 x Transaldolase TALDO1 P37837 xTranscription factor BTF3 BTF3 P20290 xTransgelin-2 (Fragment) TAGLN2 X6RJP6 xTropomyosin alpha-1 chain (Fragment) TPM1 H0YKX5 x xTubulin alpha-1B chain TUBA1B P68363 x xTubulin alpha-4A chain TUBA4A P68366 xTubulin beta chain TUBB P07437 xUbiquitin-40S ribosomal protein S27a RPS27A P62979 xUbiquitin-conjugating enzyme E2 N UBE2N F8VSD4 x Ubiquitin-like modifier-activating enzyme 1 UBA1 P22314 x xVimentin VIM P08670 x x

Homo sapiens protein sequences retrieved from UniProtKB/Swiss-Prot database, with trypsin as enzyme, maximum of one missedcleavage, fixed carbamidomethyl modification for cysteine residues,and oxidation of methionine as variable modification. Peptides andprotein tolerances were set as automatic, allowing minimumfragment ion per protein as 5, minimum fragment ion per peptideas 2, minimum peptide matches per proteins as 1 and falsediscovery rate (FDR) as 4%. Only proteins identified in two out ofthree biological replicates were considered for qualitative andquantitative analysis in order to improve confidence andreproducibility. Data sets were normalized using the “auto-normalization” function of PLGS and label-free quantitativeanalysis was performed from peak intensity measurements (Hi3method) (14) using PLGS ExpressionE algorithm. Proteins withregulation-probability (P) values below 0.05 or higher than 0.95were taken as differentially regulated between samples. Functionalannotation and enrichment analysis were performed usingPANTHER (Protein Analysis Through Evolutionary Relationships)database (15) matched with the Homo sapiens genome. The Fisher’sexact test was used with FDR correction. The plots of mostrepresentative and significant biological processes were constructedusing ggplot2 R package.

Results

A549/CDDP cells displayed clinically relevant levels of drugresistance, with an IC50 value 3.5-fold higher than that of theparental A549 cells (Figure 1A). A549/CDDP cells presentedunstable resistance, gradually resuming the resistance levelof the parental cells after ~30-45 days of cultivation in drug-free medium (data not shown). A549/CDDP cells, therefore,represent a cellular model for studying non-genetic resistanceto CDDP.

Protein dynamics induced by CDDP in A549 andA549/CDDP cells were evaluated by BONCAT and LC-MS/MS. Considering all time-points of the experiment (2, 4and 8 h), a total of 173 and 151 unique proteins wereidentified from A549 cells cultured in absence and presenceof CDDP, respectively. A total of 148 and 153 unique newlysynthesized proteins were identified in A549/CDDP cellsduring the time-course experiment in absence and presenceof CDDP, respectively. The complete lists of proteinsidentified in each experiment and sample are available athttps://zenodo.org/record/4640561#.YIhJhJAzZPY.

Initially, we compared the proteins synthesized by A549 andA549/CDDP cells in drug-free medium to identify differencesin their steady-state proteome. The complete list of proteinsdifferentially expressed between A549 and A549/CDDP cellsduring culture in drug-free medium is available athttps://zenodo.org/record/4640561#.YIhJhJAzZPY. A549 andA549/CDDP cells presented different expression profiles, withthe enrichment of Gene Ontology (GO) terms related to proteinfolding, stabilization and turnover, telomere organization andmaintenance, cellular adhesion, actin cytoskeleton, and cellularresponse to drug in A549/CDDP cells (Figure 1B). The

complete list of GO annotations is available athttps://zenodo.org/record/4640561#.YIhJhJAzZPY. Our resultssuggest that these biological processes were selected/inducedby drug treatment during the acquisition of non-geneticresistance by A549/CDDP cells.

Next, proteins synthesized by A549 and A549/CDDP cellsin the presence and absence of CDDP were compared, time-point by time-point, to identify proteome changes induced bydrug exposure. The complete lists of proteins induced byCDDP in each time-point of the experiment are available athttps:/ /zenodo.org/record/4640561#.YIhJhJAzZPY.Considering all time-points of the experiment, proteins inducedby CDDP in A549 and A549/CDDP cells are presented inFigure 2A and Table I. GO terms enriched by CDDP in eachcell are shown in Figure 2B. GO terms related to telomeremaintenance, protein folding and stabilization, cell adhesion,cytoskeleton, and cell redox homeostasis were found enrichedupon CDDP exposure in both A549 and A549/CDDP cells,while proteins involved response to drug, metabolic pathwaysand regulation of mRNA processing/splicing were induced byCDDP only in A549/CDDP cells.

Discussion

Herein, we used BONCAT and LC-MS/MS to monitor geneexpression reprogramming induced by CDDP in lung cancercells with distinct phenotypes of drug sensitivity. Our resultsrevealed that proteins involved in proteostasis, telomeremaintenance, cell adhesion, cytoskeleton remodeling and cellredox homeostasis were up-regulated by CDDP in both A549and A549/CDDP cells. These results highlight theimportance of these molecular pathways in the non-geneticadaptive response to therapy. Interestingly, the profile ofbiological processes enriched in A549 cells after CDDPtreatment is very similar to those identified in the steady-state proteome of A549/CDDP cells, which reinforces theparticipation of these molecular mechanisms in promotingcell survival during drug exposure and suggests their positiveselection in CDDP-resistant cells. It is also important to notethat the biological processes identified in our cellular modelof non-genetic resistance resemble those described in thegenetic resistance to CDDP (16, 17), which suggests thatcomparable pro-survival pathways could be activated bygenetic and epigenetic mechanisms. In this sense, it is notsurprising that most proteins up-regulated by CDDP arerelated to stress response pathways, as this signaling is ofcrucial importance in limiting cellular damage and enhancingcell survival.

Chaperones, foldases and proteases involved in the proteinquality control network were found up-regulated by CDDPtreatment in A549 and A549/CDDP cells. During stressresponse, cells use quality-control strategies to maintainprotein homeostasis (proteostasis) (18). Thus, cells with up-


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regulated quality control machinery may be more efficient tocope with protein misfolding stress caused by CDDPexposure. In fact, unfolded protein response (UPR) activationis commonly observed in cancer and correlates with drugresistance (19).

Proteins reported to be involved in telomere maintenance,such as TRiC/CCT complex (20) and hnRNPs (21), had theirexpression increased in A549 and A549/CDDP cells afterCDDP exposure. Human telomeric DNA (tandem repeats of5’-TTAGGG-3’ sequences) is a potential target for CDDP-induced cross-links (22). Cell treatment with CDDP resultsin markedly shortened telomeres, which can induce apoptosis(23, 24). Thus, enhanced mechanisms of telomeremaintenance can be involved in resistance to CDDP-inducedapoptosis.

Proteins related to cell adhesion and cytoskeletalrearrangements were found up-regulated by CDDP exposurein both A549 and A549/CDDP cells. Cell adhesion toextracellular matrix (ECM) elicits activation of different pro-survival signaling pathways which contribute to tumordevelopment and chemoresistance, in a mechanism referredas cell adhesion-mediated drug resistance (CAM-DR) (25,26). In addition, cell adhesion triggers cytoskeletonreorganization, regulating cellular stiffness (26). Cisplatinhas been reported to induce considerable remodeling of actincytoskeleton, increasing stress fibers and cell stiffness (27).CDDP-resistant cell lines showed a significantly higher cellstiffness when compared to their drug-sensitive counterparts(28). Therefore, proteins involved in cell adhesion andcytoskeletal rearrangement could be relevant targets tocounteract cellular resistance to CDDP (26, 29, 30).

The expression of detoxifying enzymes was induced byCDDP in A549 and A549/CDDP cells. CDDP generates arobust oxidative stress and, therefore, cells need to developantioxidant mechanisms to deal with drug toxicity (31). Cellswith an increased detoxification capacity have been reportedto be more chemoresistant (32). In accordance, our resultsindicate that detoxifying enzymes may play a relevant rolein non-genetic response to CDDP.

On the other hand, some biological processes, such asdrug response, metabolic pathways, and mRNA processingand splicing, were up-regulated by CDDP only inA549/CDDP cells, which suggest their relevance to thedevelopment and/or maintenance of a drug resistancephenotype.

Proteins identified in A549/CDDP cells associated to drugresponse include ALDH3A1, HMOX1 and NQO1 proteins.Aldehyde dehydrogenases are markers of cancer stem cellsand associated with cancer chemoresistance (33). HMOX1and NQO1 have cytoprotective roles and enhance resistanceto anticancer therapies (34, 35).

mRNA processing and splicing were also differentiallyrepresented in A549 and A549/CDDP cells upon CDDP

exposure. Post-transcriptional mechanisms increaseproteome diversity, enhancing tumor cell adaptation tochemotherapy (36). Therefore, it is likely that thesemechanisms play major roles in the development of non-genetic resistance to CDDP.

Regarding mechanisms of transcriptional regulation, weidentified the BTF3 transcription factor up-regulated inA549/CDDP cells after CDDP exposure. BFT3 expressionhas been associated with cancer stem cells (37), which areknown to be involved in cancer growth, metastasis andchemoresistance.

Possible differences in the metabolism of A549 andA549/CDDP cells upon CDDP exposure were also detectedby our proteomic approach, with the up-regulation ofproteins involved in glycolysis and pentose phosphatepathways in A549/CDDP cells. Metabolic reprogramming isoften associated with drug resistance, as the alteredmetabolism can confer adaptive, proliferative, and survivaladvantages in adverse conditions (38). Our results pointedthat metabolic reprogramming could also be a non-geneticresistance mechanism to CDDP.

The results presented herein shed light on the mechanismsof gene expression regulation involved in the non-geneticresistance to CDDP. Knowing these mechanisms is afundamental step for the development of novel strategies tomonitor and counteract non-genetic resistance in cancer.

Conflicts of Interest

The Authors declare that no conflicts of interest exist with regardto the present study.

Authors’ Contributions

Conceptualization: C.S.D. and K.M.M.; Methodology: C.S.D. andK.M.M; Investigation: C.S.D., C.L.M. and N.A.C.; Formal analysis:C.S.D. and K.M.M.; Visualization: C.S.D.; Project administration:K.M.M.; Funding acquisition: K.M.M.; Resources: H.B.F., A.Z. andK.M.M.; Writing – original draft: C.S.D.; Writing – review andediting: H.B.F., A.Z. and K.M.M.

Acknowledgements

This work was funded by Fundação de Amparo à Pesquisa doEstado do Rio Grande do Sul (FAPERGS), grant number 16/2551-0000 286-0 (K.M.M.). C.S.D. and C.L.M. were supported byConselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq) scholarships. N.A.C. was supported by a Coordenação deAperfeiçoamento de Pessoal de Nível Superior (CAPES)scholarship. We thank the Uniprote-MS (CBiot/UFRGS) fortechnical support with LC-MS/MS.

Supplementary Material

Available at: https://zenodo.org/record/4640561#.YIhJhJAzZPY


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Received March 26, 2021Revised April 22, 2021Accepted April 27, 2021


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proteomic analysis of the non-genetic response to

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