Analysis of TAp73-Dependent Signaling via Omics TechnologiesAngelo D’Alessandro,† Cristina Marrocco,† Sara Rinalducci,† Angelo Peschiaroli,‡ Anna Maria Timperio,†

Lucilla Bongiorno-Borbone,§ Alessandro Finazzi Agro,§ Gerry Melino,§,⊥ and Lello Zolla*,†

†Department of Ecological and Biological Sciences, University of Tuscia, Largo dell’Universita, snc, 01100 Viterbo, Italy;‡Institute of Cellular Biology and Neurobiology, CNR, Rome, Italy§Department of Experimental Medicine and Biochemical Sciences, University of Rome “Tor Vergata”, Via Montpellier 1, 00133Rome, Italy⊥Medical Research Council, Toxicology Unit, Hodgkin Building, Leicester University, Lancaster Road, P.O. Box 138, Leicester LE19HN, U.K.

*S Supporting Information

ABSTRACT: Transactivation-proficient (TA) p73 is a transcription factorbelonging to the p53 family, which regulates a variety of biological processes,including neurogenesis, differentiation, apoptosis, and DNA damagecheckpoint response. In the present study, we adopted multiple Omicsapproaches, based upon the simultaneous application of metabolomics,lipidomics, and proteomics, in order to dissect the intracellular pathwaysactivated by p73. As cellular model, we utilized a clone of the humanosteosarcoma SAOS-2 cell line that allows the expression of TAp73α in aninducible manner. We found that TAp73α promoted mitochondrial activity(accumulation of metabolic intermediates and up-regulation of proteins relatedto the Krebs cycle), boosted glutathione homeostasis, increased arginine−citrulline−NO metabolism, altered purine synthesis, and promoted thepentose phosphate pathway toward NADPH accumulation for reducing andbiosynthetic purposes. Indeed, lipid metabolism was driven toward theaccumulation and oxidation of long-chain fatty acids with pro-apoptotic potential. In parallel, the expression of TAp73α wasaccompanied by the dephosphorylation of key proteins of the mitotic spindle assembly checkpoint. In conclusion, the obtainedresults confirm existing evidence from transcriptomics analyses and suggest a role for TAp73α in the regulation of cellularmetabolism, cell survival, and cell growth.

KEYWORDS: p73, metabolomics, proteomics, lipidomics

■ INTRODUCTION

In 1997, p73 emerged as a structural and functional homologueof the tumor suppressor p53.1 Both p73 and p53 genes consist ofan N-terminal transactivation domain, a central sequence-specific DNA-binding domain, and a C-terminal oligomerizationdomain.2,3 The human p73 genes are composed of 15 exonsspanning over 80 000 bp on chromosome 1p36.3 (GenBankAccession Number: AL136528).2 In contrast to p53, the p73gene is very complex, since it expresses at least seven alternativelyspliced C-terminal isoforms (α, β, γ, δ, ε, ζ, and η, also referred toas transactivation proficient or TA variants) and at least fouralternatively spliced N-terminal isoforms initiated at differentATG codons (including ΔNp73α and ΔNp73β, lacking thetransactivation domain).2−5 Alternatively spliced TA isoformsdisplay different transcriptional and biological properties. Basedon their sequence similarity at the DNA binding domain, it is notsurprising that p53 and TAp73 isoforms can share sometranscriptional targets and, as a consequence, elicit similarbiological effects. Ectopic expression of TAp73 isoform is indeedable to transactivate p53-responsive genes causing cell cycle

arrest and apoptosis.2 TAp73-triggered apoptosis follows severalpathways,6−8 including mitochondrial intrinsic pathway(through activation of PUMA,9 Noxa,10 and Bax6,9) and viaendoplasmic reticulum (ER) stress through the induction ofScotin.11 Pro-apoptotic activity of TAp73 variants can bemodulated12,13 by phosphorylation of TAp73 via checkpointkinase Chk1 and Chk214 or AMPK15 or through collaborativebinding to c-Abl.16,17

In contrast to the TAp73 isoforms, the ΔN variants ΔNp73αand ΔNp73β, which are generated by alternative promoterutilization, exhibit dominant-negative behavior toward wild-typep73 as well as p53, display antiapoptotic and oncogenic activity,and are involved in the regulation of the DNA damagecheckpoint response.18−22 Thus, the biological outcome of p73activity is determined by the relative expression levels of itsTAp73 and dominant-negative ΔNp73 variants in cells.23

However, exceptions to this assumption are all but infrequent,

Received: June 11, 2013Published: August 7, 2013

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especially in the light of recent reports indicating a controversialrole of the TAp73α isoform in mitigating apoptotic events in K-562 leukemia cells,24 small cell lung carcinoma,25,26 and humanovarian carcinoma cell line A2780.27 Therefore, although TAp73isoforms are able to bind specifically to DNA through p53responsive elements, transcriptional targets of p73 isoforms andp53 (and thus their biological functions) can be not entirelycomparable. Accordingly, the analysis of the isoform-specific p73knockout mice revealed a peculiar function played by p73 duringneurodevelopment, mitotic spindle checkpoint, and DNAdamage checkpoint response, suggesting that p73 biologicalfunctions are not entirely redundant with those of p53.Besides activating the transcription of genes encoding

proteins, p73 is also able to upregulate the expression ofmiRNAs, such as let-7, miR-34, miR-15/16a, miR-145, miR-29,miR-26, miR-30, miR-146a,28 and miR-200 family.29 Whileconnections between miRNAs and cancer cell metabolism areincreasingly being confirmed,30,31 the actual effect of TAp73expression in cancer cell metabolism still remains under-investigated. Indeed, energy metabolism is a key hallmark ofcancer,32,33 and tumor suppressor genes such as p53 play acentral role in the modulation of glucose metabolism.34 So far,available results suggest a role for TAp73α in the deregulation ofmitochondrial energy metabolism and exacerbation of mitochon-dria-induced ROS production in HCT116 and TAp73 knock outMEF cells.35 The underlying mechanisms could involve thecomplex IV subunit cytochrome C oxidase subunit 4 (Cox4i1), adirect target of TAp73α.35

To further understand the TAp73α-mediated cellular signal-ing, we performed an integrated omics approach, based upon thesimultaneous application of metabolomics, lipidomics, proteo-mics, and phosphoproteomics. The application of multiple omicsanalyses to the same biological matrix holds several advantages,in that it helps to cope with the intrinsic limitations of eachindependent approach36 while providing further clues to theunderstanding of the biological phenomenon under investiga-tion, even when scarce direct overlap among results fromdifferent omics is observed.37 In this view, simultaneousapplication of multiple omics disciplines represents the inevitableevolution of unsupervised approaches toward the achievement ofan actual Systems Biology-wide comprehension of livingsystems.38

■ EXPERIMENTAL PROCEDURES

Cell Culture and Reagents

Human osteosarcoma SAOS-2 with doxycycline-inducibleexpression of HA-TAp73α were cultured in Dulbecco’s modifiedEagle’s and F12 medium (Gibco, Invitrogen), supplementedwith 10% fetal bovine serum (FBS), 100 μg/mL penicillin, and100 μg/mL streptomycin (all Gibco, Invitrogen) and cultured at37 °C with 5% CO2. To induce TAp73α expression, Saos-2−TAp73α cells were grown in the presence of doxycyclin (2 μg/mL) for 24 h.RNA Isolation and qRT-PCR

Total mRNA was isolated using the RNeasy mini kit (Qiagen,Duesseldorf, Germany) following manufacturer recommenda-tions. Total RNA was quantified using a NanoDrop spectopho-tometer (Thermo Scientific, Delaware, USA) and used for cDNAsynthesis using SuperScript reverse transcriptase (Promega,Fitchburg, WI, USA), according to the manufacturer’s protocol.cDNA was subsequentially used for real-time PCR analysis(qRT-PCR). Each 25 μL reaction contained 2× SYBR-Green

PCR master mix (Applied Biosystems), 0.125 μL of MultiScribereverse transcriptase (Applied Biosystems), 2 μL of cDNA, andthe appropriate specific primers (0.5 μM, sequences availableupon request). Amplification and fluorescence detectionaccording to the manufacturer’s instructions were performedusing the ABI PRISM 7700 sequence detection system (AppliedBiosystems, France). The expression of each gene was definedfrom the threshold cycle (Ct), and relative expression levels werecalculated using the 2-ΔΔCt method. The following primerswere used: forw hPGLS, 5′-CAGACCCCCCCTCCTACAG-3′;rev hPGLS, 5′-CTGCCTTGCCTTCTCCAGTT-3′; forwhp21, 5′-CACTGCCCTCCCATTACCTAG-3′, rev hp21, 5′-GAAGCTCAAGCATGGGAACAG-3′; forw hActin, 5′-GTT-GCTATCCAGGCTGTGCTA-3′; rev hActin, 5′-AATGTC-ACGCACGATTTCCCG-3′.Immunoblot Analysis, Antibodies, and Cell Cycle Analysis

Immunoblot analysis was performed using whole cell extractsobtained by lysing cell pellets with Triton buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 50 mM NaF, 1 mM EDTA, pH 8,0.1% Triton), supplemented with protease and phosphataseinhibitors. Proteins were separated by SDS-PAGE, transferredonto PVDF membranes, and blocked with PBS-T (phosphate-buffered saline and 0.1% Tween-20) containing 5% nonfat drymilk for 1 h at room temperature (RT). The incubation withprimary antibodies was performed for 2 h at RT, followed byincubation with the appropriate horseradish peroxidase-con-jugated secondary antibody. Detection was performed with ECLWestern blot reagent (Perkin-Elmer). Mouse monoclonalantibodies were from Covance (anti-HA), Sigma (actin), andSanta Cruz (anti-p21). Anti-rabbit IgG or anti-mouse IgGhorseradish peroxidase-conjugated antibodies were purchasedfrom Perkin-Elmer.Quantification of sub-G1 population and cell cycle phase was

performed by FACS analysis of propidium iodide-stained nuclei,carried out in a FACScan flow cytometer (Becton Dickinson,Heidelberg, Germany) using the CELLQuest software system.

Integrated Omics

Extended details about integrated omics analyses are provided inthe Supporting Information, Materials and Methods extended.

Untargeted Metabolomics Analyses

One million cells per group (control and p73α-induced SAOS-2cells) were exploited for subsequent metabolomics andlipidomics analyses. Samples were extracted following theprotocol byD’Alessandro et al.39 Extracted lipids andmetaboliteswere separated with an Ultimate 3000 rapid resolution HPLCsystem (LC Packings, DIONEX, Sunnyvale, CA, USA),equipped with a reprosil C18 column (2.0 mm × 150 mm, 2.5μm, Dr Maisch, Germany) for metabolite and lipid separation.Untargeted metabolomics analyses were performed throughdirect elution onto an electrospray hybrid quadrupole time-of-flight mass spectrometer, MicroTOF-Q (Bruker-Daltonik,Bremen, Germany) equipped with an ESI ion source. Owingto the use of linear ion counting for direct comparisons againstnaturally expected isotopic ratios, time-of-flight instruments aremost often the best choice for molecular formula determination.Calibration of the mass analyzer is essential in order to maintain ahigh level of mass accuracy. Instrument calibration wasperformed externally every day with a sodium formate solutionconsisting of 10 mM sodium hydroxide in 50% isopropanol/water, 0.1% formic acid. Automated internal mass scalecalibration was performed through direct automated injection

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of the calibration solution at the beginning and at the end of eachrun by a six-port divert valve.

Targeted Metabolobomics and Lipidomics: MultipleReaction Monitoring (MRM)

Metabolites and lipids of interest were thus further tested forvalidation with multiple reaction monitoring (MRM) againstexternal standards from SIGMA Aldrich (Milan, Italy), aspreviously reported.39 Metabolites and lipids were directly elutedinto a high capacity ion trap HCTplus (Bruker-Daltonik,Bremen, Germany), and runs were acquired in positive andnegative ion mode, as previously described.39 Triplicate runs foreach group (TAp73α-induced vs noninduced controls) wereexported as mzXML files and processed through MAVEN.40

Mass spectrometry chromatograms were elaborated for peakalignment, matching, and comparison of parent and fragmentions and tentative metabolite identification (within a 20 ppmmass-deviation range between observed and expected resultsagainst the KEGG pathway database41). Relative quantitationand pathway representations were determined upon normal-ization against noninduced controls in a pathway-wise fashion.Data were further refined and plotted with GraphPad Prism 5.0(GraphPad Software Inc.).

Proteomics Analyses

Protein extraction and proteomics analyses were performed aspreviously reported.42 A total of eight 2DE gels have beenperformed during the proteomic analysis of membrane proteins(four biological replicates per group, TAp73α-induced andnoninduced controls). For 2DE analysis, cellular membraneprotein extracts were prepared as previously described, withminor modifications.42

Proteins were visualized through sensitive Coomassie BrilliantBlue G-250 stain.43

Stained gels were digitalized, and image analysis wasperformed using the Progenesis SameSpot softwarev.2.0.2733.19819 software package (Nonlinear Dynamics, NewCastle, U.K.).Spots from 2-DE maps of biological interest (p < 0.05

ANOVA) were carefully excised from the gel and subjected to in-gel trypsin digestion according to Shevchenko et al.44 with minormodifications.TiO2 enrichment and CID/ETD-analysis of phosphopeptides

were performed as previously reported.45−47 To provide a largerlist of phosphorylation sites, the TiO2-enriched samples wereanalyzed using a split-free nanoflow liquid chromatographysystem (EASY-nLC II, Proxeon, Odense, Denmark) coupled to a3D-ion trap (model AmaZon ETD, Bruker Daltonik, Germany)equipped with an online ESI nanosprayer. A detailed descriptionof the ETD setup of the ion trap instrument including the

generation of the reagent anion of fluoranthene was givenpreviously.48 The following parameters were adopted fordatabase searches with Mascot search program (in-house version2.2, Matrix Science, London, U.K.): NCBInr database (releasedate March 10, 2013; 247 209 sequences); taxonomy=Homosapiens; peptide mass tolerance of ±0.3 Da; fragment masstolerance of ±0.3 for CID ions and of ±1.3 Da for ETD ions;enzyme specificity trypsin with two missed cleavages considered;fixed modifications, carbamidomethyl (C); variable modifica-tions, oxidation (M), phosphorylation (STY). Phosphopeptideidentifications were accepted if the Mascot score was over the95% confidence limit based on the “identity” score of eachpeptide. A delta ion score was calculated of all phosphopeptidescontaining more than one serine, threonine, or tyrosine residueby taking the difference between the two top ranking Mascot ionscores. Phosphorylation site assignments with a delta score >5were automatically accepted.49

■ RESULTS AND DISCUSSION

In order to evaluate the TAp73-dependent signaling, we utilizeda subclone of the human osteosarcoma SAOS-2 cell line (p53null cells) where the exogenous expression of HA-TAp73α isunder control of a doxycycline responsive promoter. Upon 24 hof doxycycline treatment, the expression of TAp73α and itstranscriptional target gene p21 is increased. The ectopicexpression of TAp73α is able to induce cell cycle arrest orapoptosis within 48 h from doxycyline induction.9 Since in thepresent study we were interested in investigating the eventsleading to TAp73α-driven apoptosis, we analyzed the cell cycleprofile of the cells 24 h from TAp73α induction. We confirmedthat this time frame is adequate for further omics analyses in thatearly evidence of cell cycle arrest in G2/M phase and slightincreases in the percentage of apoptotic cells were already visibleat 24 h from the induction (Figure 1).

Proteomics Analyses

Proteomics analyses revealed that the induction of TAp73αexpression resulted in the up-regulation of 15 and down-regulation of 9 protein spots. Results are reported in Table 1,along with the indication of the extended protein name,theoretical MW, and pI’s, gene identifiers (NCBI gi), andidentification details (the number of unique peptides, sequencecoverage, and MASCOT scores), other than the relative fold-change variation from 2DE analyses. Extended data about MS/MS-based identifications are provided in Supplementary Table 1and Mascot Search Results, Supporting Information. Distribu-tion of protein spots in the control (left panel) and in theTAp73α-induced (right panel) SAOS-2 cells is showed on 2DEmaps in Figure 2.

Figure 1. (A) Human osteosarcoma SAOS-2 cells (SAOS-2−TAp73α) with doxycycline-inducible expression of HA-TAp73α were grown in thepresence of doxycyclin (2 μg/mL) for 24 h. Whole cell extracts of untreated and treated cells were utilized for Western blot analysis using the antibodiesto the indicated proteins. (B) SAOS-2−TAp73α treated as in panel Awere subjected to FACS analysis. The percentage of sub-G1, G1, S, andG2/M cellsis reported.

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First of all, it is worthwhile noting that we assayed cells at 24 hafter induction of TAp73α. This time frame is indeed sufficient toobserve the induction of direct target genes of TAp73transcriptional activity (Figure 1A) and early biological responses

(such as cell cycle arrest, as reported above, Figure 1B). SinceTAp73α is expressed roughly already at 8−12 h from inductionin the Tet-On doxycycline-inducible SAOS2 cell line,11 some ofthe observed changes at the proteomics level might be also

Table 1. Mass Spectrometry-Based Identification of Differentially Espressed Proteins upon TAp73α Induction

spotno. protein ID

Mr (Da) theor/exptl

pI theor/exptl NCBI gi no.

no. of uniquepeptidesa

sequencecoverage %

Mascot score(total)

foldchange

Overexpressed in SAOS-2 TAp73α Induced230 90 kDa heat shock protein 83584/85600 4.97/4.85 gi|225608 6 (5) 9 226 3494 45 kDa calcium-binding protein 41895/55800 4.76/4.5 gi|18699732 2 (1) 5 92 3.5

coiled-coil domain containing 97 39265/55800 4.52/4.5 gi|16418351 2 (2) 7 90mitochondrial dihydrolipoamidesuccinyltransferase

49000/55800 9.01/4.5 gi|499719 2 (1) 4 84

UV excision repair protein RAD23 homologueA variant

41402/55800 4.66/4.5 gi|62089006 2 (2) 4 55

591 calumenin 37164/50000 4.47/4.3 gi|2809324 4 (4) 16 141 1.5859 splicing factor, arginine/serine-rich 1 32321/36150 10.37/5.3 gi|

1196148945 (3) 17 198 1.8

874 microtubule-associated protein RP/EB familymember 1

30151/35600 5.02/5.0 gi|6912494 4 (3) 16 162 1.5

tubulin folding cofactor B 27663/35600 5.14/5.0 gi|2343185 5 (3) 21 91909 coatomer protein complex, subunit epsilon 34688/34300 4.97/5.0 gi|

1196051626 (5) 20 158 1.6

938 14-3-3 protein epsilon 29326/31300 4.63/4.6 gi|5803225 18 (16) 70 2100 1.7975 14-3-3 protein epsilon 29326/31300 4.63/4.6 gi|5803225 2 (2) 10 240 1.8987 prohibitin 29843/30000 5.57/5.7 gi|4505773 5 (5) 23 181 1.5

proteasome beta 7 subunit 30260/30000 7.57/5.7 gi|62898273 3 (2) 8 916-phosphogluconolactonase 27815/30000 5.70/5.7 gi|

1196050202 (1) 7 80

1118 HSPC029 25329/25800 4.89/4.7 gi|5114051 3 (3) 17 266 1.6proteasome subunit Y 25527/25800 4.80/4.7 gi|558528 4 (4) 16 141

1324 RNA polymerase II subunit 17203/17500 4.5/4.3 gi|1017823 3 (3) 20 236 1.51439 PSMC3 45507/49900 5.39/5.1 gi|48145579 14 (11) 36 547 1.6

α-tubulin 50810/49900 5.02/5.1 gi|37492 2 (2) 6 2001454 mitochondrial ATP synthase, H+ transporting

F1 complex β subunit48083/50000 4.95/5.0 gi|89574029 8 (6) 23 567 2

β-tubulin 50240/50000 4.75/5.0 gi|338695 4 (2) 10 147p48 41470/50000 5.09/5.0 gi|904032 3 (2) 7 130

1460 smooth muscle myosin alkali light chain(myosin light polypeptide 6)

17772/17000 4.57/4.2 gi|467828 2 (2) 15 112 1.7

1478 protein disulfide isomerase-related protein 5 46512/50200 4.95/5.1 gi|1710248 18 (11) 41 693 1.7dynactin subunit 2 44906/50200 5.06/5.1 gi|5453629 13 (7) 32 537ATP synthase subunit β, mitochondrialprecursor

56525/50200 5.26/5.1 gi|32189394 7 (7) 19 534

α-tubulin 50788/50200 4.94/5.1 gi|158259731

8 (5) 23 322

Tat binding protein 1, TBP-1 = transcriptionalactivator

49322/50200 5.08/5.1 gi|263098 10 (5) 21 258

p48 41470/50200 5.09/5.1 gi|904032 3 (3) 9 156Tat binding protein 7, TBP-7 = transcriptionalactivator

51633/50200 5.52/5.1 gi|263099 7 (2) 12 90

Overexpressed in SAOS-2 TAp73α Noninduced Controls387 plastin-3 isoform 1 71279/79000 5.41/5.75 gi|7549809 22 (18) 34 1022 1.5489 α-tubulin 50804/51000 4.94/5.1 gi|34740335 18 (14) 43 998 1.5

vimentin 53738/51000 5.03/5.1 gi|340219 9 (6) 20 302596 chaperonin (HSP60) 61157/61900 5.70/5.7 gi|306890 2 (1) 5 72 1.7638 actin 42480/48000 5.23/5.1 gi|178027 3 (2) 8 93 1.7649 deaminase a, adenosine 41009/48000 5.62/5.7 gi|224877 5 (4) 15 227 1.7998 prosomal RNA-binding protein p27K 27838/31900 6.34/6.3 gi|446773 12 (10) 43 1082 1.7

enoyl-CoA hydratase 31807/31900 8.34/6.3 gi|1922287 8 (7) 33 6041199 not identified 1.91231 ribosomal protein L12 17979/19000 9.48/5.1 gi|4506597 2 (2) 14 94 1.51441 mitochondrial ribosomal protein L7/L12 21593/21000 9.01/5.3 gi|1313962 4 (4) 16 154 1.8

aIon score cutoff = 30. Values in parentheses indicate statistically significant peptides (p < 0.05). Only proteins with at least two unique peptides areconsidered and reported.

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attributable to early indirect transcriptional effects of TAp73αactivation.Differentially expressed proteins could be further divided on

the basis of their biological activity, including (i) proteinsinvolved in p73 protein stabilization and in the regulation of itsactivity, (ii) proteins involved in p73 degradation and proteinturnover, (iii) proteins reflecting an active transcriptional stateupon TAp73α-induction, and (iv) proteins linked tometabolism.Regulation of p73 Activity.We found that the 90 kDa heat

shock protein (HSP90, spot no. 230, Table 1) is up-regulated inTAp73α-induced cells. This phenomenon might be related tothe stabilization of the p73 protein through a mechanismconserved within the p53/p63/p73 protein family. Indeed,accumulation of the p73-homologue p53 results from ATMkinase-mediated phosphorylation of p53 at serine 15 or byHSP90-binding to wild-type p53.50 Inhibition of HSP90−p53interaction by geldanamycin completely prevented p53 accumu-lation in ATM-deficient cells.50 Analogous mechanisms couldcontribute to the stabilization and accumulation of newlyexpressed TAp73α.Another protein whose expression is increased in response to

TAp73α induction is 14-3-3 protein ε (spot nos. 938 and 975,Table 1). Several publications suggest that activation of p53following DNA damage directly up-regulates the levels 14-3-3σand that this process is critical for the regulation of the G2/Mcheckpoint.51,52 The interactions between p53 and 14-3-3 arecomplex and are likely to be at least partially cell type specific. Forexample, 14-3-3σ and other 14-3-3 isotypes, including 14-3-3εhave been reported to directly bind to p53 itself,52,53 increasingthe DNA-binding function and activity of p53. However, p53activation depends upon dephosphorylation and binding to 14-3-3 proteins.52 Again, the up-regulation of 14-3-3 protein ε inTAp73α-induced cells is suggestive of further shared similaritiesbetween p53 and p73 activation cascades.Protein Degradation and ER Stress. Protein degradation

determines the outcome of many cellular physiologicalprocesses. Degradation of proteins by the proteasomes occursvia various and tightly regulated pathways, the most extensivelystudied one being the ubiquitin−26S proteasome pathway.54

The proteasomal degradation of the tumor suppressor p73 isregulated by both polyubiquitination (a mechanism that isinhibited by the promyelocytic leukemia (PML) protein55,56)and by a ubiquitin-independent process.57,58 Unlike p53, p73 istargeted for ubiquitination by another E3 ubiquitin ligase thanMDM2.59,60

Hereby, we could find an up-regulation of proteasome 26S(spot no. 1439) and TAT-binding protein 1 (an ATPase of 19Sregulatory particles of the 26S proteasome,61 spot no. 1478)upon induction of the expression of TAp73α (Table 1). Inaddition, we could observe up-regulation of spot no. 987 in theTAp73α-induced group, also identified as proteasome 20Ssubunit beta 7 (Table 1). Overall, these findings are suggestive ofa likely protein turnover upon p73 induction, a process thatmight result in a negative feedback loop toward homeostasis ofthe levels of p73 protein itself. Another tentative explanationstems from increased rates of protein degradation through theproteasome system under conditions of endoplasmic reticulum(ER) stress,62,63 a condition that is known to be induced byTAp73α through a Scotin-mediated pathway.11 Misfolding ofproteins is associated with ER stress,62,63 hereby indirectlydocumented by the overexpression of protein disulfide isomerase5 (PDIA5, spot no. 1478 in Table 1) in TAp73α-induced cells.Indeed, PDIA5 is an ER enzyme that catalyzes the rearrangementof disulfide bonds between cysteine residues within proteinsduring folding. This allows proteins to quickly find the correctarrangement of disulfide bonds in their fully folded state, whichmakes PDIA5 a catalyst of proper protein folding that respondsto ER stress stimuli.64

In line with the posited hypothesis about proteomics changesreflecting the likely involvement of p73α in ER stress, the p73-induced up-regulation of protein UV excision repair proteinRAD23 homologue A (spot no. 494, Table 1) can be related toDNA damage responses, ER stress, and protein degradation.65

The DNA repair protein RAD23 has been shown to inhibitdegradation of specific substrates in response to DNA damage,including the associated nucleotide excision repair protein XPC,thereby stimulating DNA repair in a mouse model.65 TheRAD23 homologue MIF1 binds to the 19S regulatory subunit ofproteasome 26S and could play a role in the translocation of the26S proteasome toward the ER membrane, thereby enhancingER-associated protein degradation.65 It is at least worth notingthat another protein was identified under the spot no. 494,namely 45 kDa calcium-binding protein, a protein that isinvolved in the regulation of calcium-dependent activities in theendoplasmic reticulum lumen or post-ER compartment.66

Consistently, ER stress induced by p73 proteins follows aScotin/caspase-dependent pathway.9,11,17,67,68

Further clues to the p73α involvement in DNA damage repairand proteasome-dependent degradation stem from differentialphosphoproteomics results (Table 2).

Figure 2. 2DE maps of noninduced controls (left panel) versus TAp73α-induced SAOS-2 cells. Spot numbers indicate differentially expressed proteins(p < 0.05 ANOVA) upon statistical analyses through Progenesis Samespots and relate to protein identifications reported in Table 1.

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Phosphorylation of the ubiquitin fusion-degradation likeprotein at Ser299 (uniquely found in the TAp73α expressingcells,Table 2) is associated with inactivation of the pathwaysleading to DNA damage repair and mTOR signaling occurringduring mitosis or in response to ATM activation.69 Connectionsbetween themTOR−p73 signaling axis are not novel.70−72 In thepresent study, additional supporting evidence is provided byphosphorylation at Ser250 of the proteasome subunit alpha type-3 (only present in the noninduced cells, Table 2), a post-translational modification (PTM) that triggers inhibition ofproteasome activity in response mTOR73 andMAPK74 signaling.Analogous considerations can be made for microtubule

associated protein 4, phosphorylated at Ser280 in noninducedcells (Table 2), which is associated with cytokinesis duringmitosis.75 On the other hand, the up-regulation of microtubule-associated protein RP/EB family member 1 (MAPRE1, up-regulated in TAp73α-induced cells, spot no. 874 in Table 1) wasunexpected, since both p53 and p73β have been reported toinhibit transcription of this protein.76 This might reflectdifferential p73 isoform-specific downstream regulation of targetgenes or rather differential PTM to this protein upon TAp73αinduction, causing spot displacement on the 2D map at the pI-level. Indeed, most of the promitotic protein−proteininteractions are modulated by phosphorylations. For example,MAPRE1 is known to interact with the stromal interactionmolecule 1 (GOK), targeting to the growing microtubule plusends77 and promoting mitosis.75 GOK is a Ca2+ sensor thatresides in the ER, and upon Ca2+ depletion, it migrates to themembrane to trigger calcium release. Phosphorylation at Ser257of GOK (Table 2) was peculiar to the noninduced group.

Transcription Regulation by TAp73. The quality of theproteomics results is also confirmed by evidence supporting therole of p73 as a transcriptional activator. This assumption is alsounderpinned by increased levels of RNA pol II in TAp73α-induced SAOS-2 cells (spot no. 1324, Table 1).We also observed differential expression of splicing factors,

including serine/arginine-rich splicing factor 1 isoform 1 (spotn-. 859 in TAp73α-induced cells, Table 1; Table 2).78 Of note,splicing factors might regulate apoptosis through alternativeprocessing of pre-mRNA of apoptotic players.79

Other than modulating transcription and mRNA splicing,TAp73α appeared to modulate also protein translation. Wefound indeed the up-regulation of eIF-3 (HSPC029 in spot no.1118 in Table 1), a eukaryotic initiation factor that associateswith the 40S ribosome and facilitates the recruitment of eIF-1,eIF-1A, eIF-2/GTP/methionyl-tRNAi, and eIF-5 to form the43S preinitiation complex (43S PIC). The eIF-3 complexstimulates mRNA recruitment to the 43S PIC and scanning ofthe mRNA for AUG recognition.80 Mitogenic stimulation leadsto mTOR and RPTOR-dependent phosphorylation and releaseof RPS6KB1 and binding of EIF4B to eIF-3.81 However,phosphorylations to Ser133 of human elongation factor-1-δ andto Ser214 of translation initiation factor IF2 (EIF1D and eiF5B,respectively, only detected in noninduced controls, Table 2) arenecessary for human spindle assembly at mitotic checkpoints81,82

and are mTOR-dependent.73 Also, phosphorylation of humanMCM2 (only present in noninduced cells, Table 2) to Ser139,downstream of mTOR signaling,73 is pivotal in Cdc7/Dbf4-mediated phosphorylation of MCM2 in the initiation of DNAreplication.83

Together with other differential phosphorylation eventsmentioned above, since all of the observed differentialphosphorylations present in the noninduced controls and absentin TAp73α-induced cells had already been reported to bedependent on mTOR activation,73 we could conclude thatTAp73α might trigger activation of specific phosphatasesupstream of mTOR signaling. Since PI3K/Akt/mTOR signalingis important to interconnect cell growth with the regulation ofthe cellular metabolism, we decided to perform extensivemetabolomics analyses and correlate it with proteomics results.

Metabolomic Analysis

Untargeted metabolomics results are plotted in Figure 3, as fold-change variations of metabolites upon induction of TAp73αexpression, normalized to noninduced controls. Metabolites are

Table 2. Phosphopeptidomics Analyses of TAp73α-InducedSAOS-2 Cells

Phosphopeptides Unique to TAp73α-Inducedubiquitin fusion-degradation 1 likeprotein

m/z 689.2604; FVAFSGEGQSLR + Phospho(ST); Mascot score 47, NL

Peptides Unique to Noninduced ControlsGOK m/z 703.2812; AEQSLHDLQER+ Phospho (ST);

Mascot score 46, ETDhuman elongationfactor-1-δ

m/z 501.8514; ATAPQTQHVSPMR + Phospho(ST); Mascot score 45, ETD

huMCM2 m/z 658.5643; GLLYDSDEEDEERPAR +Phospho (ST); Mascot score 51, ETD

microtubule-associatedprotein 4

m/z 814.4000; DMESPTKLDVTLAK + Phospho(ST); Mascot score 53, NL

proteasome subunit alphatype-3

m/z 868.2320; ESLKEEDESDDDNM.- +Phospho (ST); Mascot score 53, ETD

translation initiationfactor IF2

m/z 705.2633; NKPGPNIESGNEDDDASFK +Phospho (ST); Mascot score 41, ETD

uncharacterized proteinC12orf4

m/z 893.0097;DAEASLAAVKSGEVDLHQLASTWAK +Phospho (ST); Mascot score 43, ETD

Shared Phosphopeptides60S acidic ribosomalprotein P1

m/z 1023.2906; KEESEESDDDMGFGLFD.- +Oxidation (M); Phospho (ST); Mascot score 69,NL

calnexin m/z 754.7977; AEEDEILNRSPR + Phospho (ST);Mascot score 74, ETD

eIF-3 p110 subunit m/z 584.8919; QPLLLSEDEEDTKR + Phospho(ST); Mascot score 76, ETD

hCG1817324, isoformCRA_a

m/z 579.5674; ASMSGELRSSGNQR + Oxidation(M); 3 Phospho (ST); Mascot score 47, ETD

hematological andneurological expressed1 protein

m/z 569.2384; RNSSEASSGDFLDLK + Phospho(ST); Mascot score 88, ETD

hepatoma-derived growthfactor

m/z 502.5566; AGDLLEDSPKRPK + Phospho(ST); Mascot score 74, NL; 67, ETD

heterogeneous nuclearribonucleoprotein D0B

m/z 684.5732; IDASKNEEDEGHSNSSPR +Phospho (ST); Mascot score 120, ETD

heterogeneous nuclearribonucleoprotein H

m/z 588.8542; HTGPNSPDTANDGFVR +Phospho (ST); Mascot score 109, ETD

HSPC121 m/z 795.3309; WLDESDAEMELR + Oxidation(M); Phospho (ST); Mascot score 40, ETD

m/z 795.2997; WLDESDAEMELR + Oxidation(M); Phospho (ST); Mascot score 46, NL

HSR1 protein m/z 544.9510; REEQTDTSDGESVTHHIR +Phospho (ST); Mascot score 82, ETD

nucleolin m/z 492.2147; LELQGPRGSPNAR + Phospho(ST); Mascot score 68, ETD

protein transport proteinSec61 subunit beta

m/z 617.2425; PGPTPSGTNVGSSGRSPSK +Phospho (ST); Mascot score 90, ETD

ribonucleoprotein La m/z 602.2233; FASDDEHDEHDENGATGPVKR+ Phospho (ST); Mascot score 94, ETD

SEPT2 protein m/z 629.9698; IYHLPDAESDEDEDFKEQTR +Phospho (ST); Mascot score 61, ETD

SEPT9 protein m/z 445.2013; HVDSLSQRSPK + Phospho (ST);Mascot score 55, ETD

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grouped following a pathway-based criterion, including (i)glycolysis (Figure 3A), (ii) Krebs cycle (Figure 3B), (iii)glutathione (GSH) homeostasis (Figure 3C), (iv) pentosephosphate pathway (PPP) (Figure 3D), (v) purine metabolism

and other signaling (Figure 3E), and (vi) arginine−citrulline−NO metabolism (Figure 3F). Results were also plotted as ametabolic interaction network on the basis of whole metabolomemaps from the KEGG pathway.41 In detail, in Supplementary

Figure 3. Pathway-wise plotting of untargeted metabolomics results, including glycolysis (A), Krebs cycle (B), GSH homeostasis (C), pentosephosphate pathway (D), purine metabolism and other signaling (E), and arginine−citrulline−NOmetabolism (F). Results are plotted as mean of valuesdetected for each metabolite in TAp73α-induced cells upon normalization to noninduced controls as means ± SEM. Asterisks indicate statisticalsignificance (p-value < 0.05 T test of induced vs noninduced control values and fold-change ≥|2|).

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Figures 1 and 2, Supporting Information, we reported themetabolome maps of noninduced cells and TAp73α-inducedcells, where metabolites are displayed as nodes (node sizesincrease and decrease in TAp73α with respect to noninducedcontrols), while directed edges indicate pathway flux directions.PIP2 and PIP3, IP3, and cAMP. On the basis of the

assumption of a likely role of the PI3K/Akt/mTOR signaling inTAp73α-modulated events, we assayed the levels of phosphati-dylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)P3 or PIP3) andbiphosphate product PIP2 (PtdIns(4,5)P2) (Figure 3E).Consistent with our hypothesis, induction of TAp73α expressionresulted in the accumulation of PIP2 and decreased levels of PIP3(purine metabolism and other signaling graph, Figure 3E).Myo-inositol (1,4,5) triphosphate (IP3, Figure 3E) is a second

messenger produced primarily in response to the stimulation ofG-protein-coupled receptor or receptor tyrosine kinases. IP3 acts

on the IP3 receptor (IP3R), an ER Ca2+ release channel thattransmits Ca2+ spikes generated by the ER to mitochondria.84 Inparticular, IP3-mediated Ca2+ signaling is critical for starvation-induced autophagy stimulation.84,85 The increase of IP3 levels inTAp73α-induced cells (Figure 3E) might stem from crosstalkbetween pathways downstream to p73 activation either leadingto apoptosis or autophagy through a delicate balance.85,86

We also observed decreases of the cAMP levels uponinduction of the expression of TAp73α (Figure 3E), an eventthat can be suggestive of a metabolic switch leading todemodulation of the cAMP-mediated activation of PKA, whichis known to result in the inhibition of p73 protein activity.87

Krebs Cycle Overactivation. It has been recentlydemonstrated that TAp73 yields the activation of mitochondrialmetabolism, ultimately resulting in ROS production35 andrerouting of cancer metabolic fluxes away from the so-called

Figure 4. A detail of the results frommultiple reaction monitoring (MRM) analyses of NADPH (A) and 6-phosphogluconic acid (B). Graphpad resultsare plotted as fold change variation in TAp73α-induced SAOS-2 cells normalized to noninduced controls. In the right panel, a detail of the extracted ionchromatograms (EIC) for the selected metabolites. (C) SAOS-2−TAp73α were treated with doxycyclin (2 μg/mL) for 24 h, and total RNA wasextracted and utilized for reverse transcription and quantitative real-time PCR (qRT-PCR) using specific primers for human PGLS2, p21, and β-actin(for quantity normalization). Results are shown as mean ± SD of three independent experiments.

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Warburg effect.88 Accordingly, we found an accumulation of theKrebs cycle intemediates (citrate/isocitrate, succinyl-coA,succinate, malate, fumarate and FADH2) in TAp73α-inducedSAOS-2 cells (Figure 3B). These metabolic changes weresupported at the protein level by the TAp73α-dependent up-regulation of the Krebs cycle enzyme mitochondrial dihydroli-poamide succinyltransferase (spot no. 494), ATP synthasesubunit beta (spot nos. 1454 and 1478 in Table 1),35 andprohibitin (spot no. 987, Table 1), a regulator of themitochondrial respiration activity.89 Prohibitin expression isinduced by metabolic stress caused by an imbalance in thesynthesis of mitochondrial- and nuclear-encoded mitochondrialproteins.89 Indirect evidence about mitochondrial transcriptionunbalance derives from the TAp73α-induced down-modulationof the mitochondrial ribosomal protein L12 (spot nos. 1231 and1441, Table 1), which is known to selectively associate withhuman mitochondrial RNA polymerase to activate tran-scription.90

Shift of Glycolysis toward PhosphoenolpyruvateAccumulation and Serine Biosynthesis. While earlyglycolysis intermediates did not show any major fluctuations,we observed that TAp73 expression induces the accumulation ofphosphoenolpyruvate (PEP, Figure 3A). Conversion ofphosphoenolpyruvate to pyruvate and generation of ATP bypyruvate kinase represents a rate-limiting step in glycolysis.Embryonic cells and tumor cells predominantly express a splicingvariant of pyruvate kinase found in muscle, PKM2.91 PKM2 cellsmaintain higher flux to the serine synthetic pathway.92 Indeed,glycolytic intermediates (especially phosphoglycerate and PEP)derived from enhanced glycolysis in cancer cells can be shuntedto generate nonessential amino acids, such as serine.91 Inagreement, we detected higher levels of serine in TAp73expressing cells (purine metabolism and other signaling, Figure3E). Notably, PKM2 is reported among the p53/p63/p73 familytargets in the p53FamTag database.93

Enhanced mitochondrial activity described above resulted inthe likely accumulation of ROS, as reflected by increased levels oflactoyl-glutathione (Figure 3A) and alteration of GSH homeo-stasis.Glutathione Homeostasis. In like fashion to p53,94,95

TAp73 is able to drive the expression of glutaminase type 2(GLS2), acting on specific p53 responsive elements present onits promoter (Velletri et al., submitted). GLS2 catalyzes theconversion of glutamine to glutamate and boosts GSHhomeostasis by triggering accumulation of both reduced(GSH) and oxidized forms of glutathione (GSSG).94,95 Sincethe levels of α-ketoglutarate did not substantially vary uponTAp73α expression (Figure 3B), we could assume that glutamine(i) might enter the Krebs cycle (which was indeed overactivated,previous paragraph) and (ii) might serve as a substrate for GSHmetabolism. Consistent with these assumptions, we detected up-regulation of all the aforementioned metabolites (glutamine,glutamate, GSH, and GSSG) and of other molecules indirectlyinvolved in GSH metabolism as they promote cysteinebiosynthesis (adenosyl-homocysteine and adenosyl-methionine)(Figure 3C). In Supplementary Figure 3, Supporting Informa-tion, we provide details of the extracted ion chromatogram (EIC)for GSSG in TAp73α-induced and noninduced cells.Pentose Phosphate Pathway. Reduction of GSSG to GSH

to reload the antioxidant battery requires the reduced coenzymeNADPH. NADPH is largely produced from NADP+ through thePPP. However, NADPH also serves as a key coenzyme inanabolic pathways, including fatty acid biosynthesis, which is

required for actively proliferating cells. In this view, p53 exerts itstumor suppressor activity also by depressing NADPHproduction through direct binding to and inhibition of the firstand rate limiting enzyme of the PPP, glucose 6-phosphatedehydrogenase.96 Alternatively, p53 is known to boost the PPPvia the up-regulation of TIGAR, which in turn results in areduced flux through glycolysis and an increase in the rates ofPPP.34 In the present study, TAp73α seems to promote PPPactivity. Indeed, we found an accumulation of the oxidative phaseintermediates of the PPP in TAp73α expressing cells (6-phosphogluconolactone, 6PGL; 6-phosphogluconic acid, 6PG;NADP+ and NADPH, Figure 3D). Untargeted metabolomicsrevealed up-regulation of the whole PPP (Supplementary Figure4, Supporting Information). In order to validate these results, weperformed a targeted metabolomics approach through MRManalyses. In Figure 4, we report the MRM spectra for NADPH(Figure 4A, upper row) and 6-phosphogluconic acid (Figure 4B,lower row). At the protein level, expression of TAp73α resultedin the up-regulation of 6-phosphogluconolactonase (PGLS, spotno. 987, Table 1), the PPP enzyme that converts 6-phosphogluconolactone to 6-phosphogluconate. This was nottotally unexpected, since in silico prediction of p53/p63/p73family-RE through p53FamTag93 indicated the presence of twoREs in the promoter region of the PGLS gene, suggesting apotential transcriptional regulation of this enzyme by TAp73. Toconfirm this assumption, we measured PGLS mRNA levels incontrol and in TAp73α expressing cells by qRT-PCR, and wefound that PGLS2 transcript levels are indeed increased inTAp73α-induced cells (Figure 4C).

Purine Metabolism. With respect to purine metabolism,TAp73α shows opposite transcriptional behavior in comparisonto p53 and the TAp73β isoform. Indeed, while p53 and p73βdown-regulate the expression of adenosine deaminase (ADA),76

TAp73α promotes ADA expression,97 although ADA proteinlevels might not proportionally increase (spot no. 649, Table 1).The ADA enzyme is involved in nucleotide metabolism, thedeficit of which causes the inhibition of DNA synthesis andrepair. ADA irreversibly deaminates adenosine, converting it tothe related nucleoside inosine by the substitution of the aminogroup for a hydroxyl group. Consistently, we found an increase ofboth inosine and adenosine upon induction of TAp73αexpression (Figure 3E).

Arginine−Citrulline−NOMetabolism. Nitric oxide (NO)is synthesized from arginine and O2 by NO synthase (NOS).Citrulline formed as a byproduct of the NOS reaction can berecycled to arginine by argininosuccinate synthetase (AS) andargininosuccinate lyase (AL). AS and sometimes AL are co-induced with inducible NOS (iNOS) in several cell lines,98

whereby NO was synthesized from citrulline (via arginine) aswell as from arginine, indicating the activation of the citrulline−NO cycle.98 As a result, high concentrations of NO (and NO-derived reactive nitrogen species, RNS) induce apoptosis byactivating the p53-dependent pathway (and possibly the p73-dependent pathway), while lower concentrations of NO induceapoptosis by ER stress.98 In the present study, we foundaccumulation of all the metabolites involved in the arginine−citrulline−NOmetabolism (arginine, argino-succinate, citrulline,ornithine, Figure 3F).

Lipidomics

Finally, we investigated the TAp73α-mediated modulation ofintracellular lipid content by analyzing the lipidic fraction ofmetabolic extracts and performing targeted MRM analyses on a

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shortlist of selected lipids. The accumulation of NADPHobserved in the TAp73α expressing cells should result in theaccumulation of long chain fatty acids, in light also of theevidence that p53/p63/p73 family members induce theexpression of fatty acid synthase (FASN), a key enzyme involvedin the biogenesis of membrane lipids.99 In particular, the ectopicexpression of TAp73α and ΔNp63α leads to an increase in themRNA levels of FASN.100 Confirmatory hints were obtainedfrom untargeted metabolomics when biosynthetic pathways forlong chain saturated and unsaturated fatty acids were considered(Supplementary Figures 5 and 6, Supporting Information).Through the analysis of the lipidic fraction of metabolic extracts,we confirmed the presence of higher levels of long chain fattyacids in TAp73α-induced cells in comparison to noninducedcells (Figure 5).

At the protein level, we found that TAp73α induction isparalleled by a decrease of enoyl-CoA hydratase (spot no. 998,Table 1). This enzyme is involved in the beta-oxidationpathway,101 by hydrating the double bond between the secondand third carbons on acyl-CoA.Taken together, these results indicate that the expression of

TAp73αmight influence lipid biosynthesis and lipid oxidation toa certain extent in the absence of external stimuli. InSupplementary Figure 7, Supporting Information, for example,we showed the differential EICs of the polyunsaturated7,10,13,16-docosatetraenoic acid, a polyunsaturated fatty acidthat acts as a potent apoptotic inducer.102 Likewise, differentialexpression of ceramide 22:0 (Figure 5) upon expression ofTAp73αmight be suggestive of increased lipid-mediated growtharrest and apoptosis signaling.103−105

Furthermore, the concept about the role of lipid oxidation(especially of γ-linolenic acid, arachidonic acid, eicosapentaenoicacid ,and decosahexaenoic acid) in the promotion of cell deaththrough a mechanism mediated by the cytochrome p450,cyclooxygenase (COX) and lipoxygenase (LOX)-dependentoxidation of long-chain fatty acids to prostaglandins, thrombox-anes, and leukotrienes is not novel.42,106 In order to test thishypothesis, we investigated the accumulation of these oxidized

lipid species and reported the TAp73α-dependent accumulationof prostaglandins (B1, D3/E3, F1α), leukotrienes (A4, B4, C4,D4, F4), and some oxy-sterols (cholest-5-ene-3β,26-diol)(Figure 6).

■ CONCLUSIONSThe simultaneous application of multiple omics approacheshelped us to collect enough pieces from the jigsaw puzzle ofTAp73α downstream activated pathways to allow us to suggestbiological interpretations, which will deserve further ad hocinvestigations to collect the missing pieces through targetedapproaches in the near future (Supplementary Figure 8,Supporting Information).From the present study, a role emerged for TAp73α at the

crossroads between prosurvival autophagic signals and ERstress/mitochondrial proapoptotic pathways. Biologically, theseopposite signals balance each other resulting in cell cycle arrestwithin 24 h from the induction of TAp73α.Several lines of evidence underpin this statement, both at the

protein and at the metabolic level, which integrate comple-mentary evidence already available from the literature.First of all, expression of TAp73α in SAOS-2 cells results in the

stabilization, accumulation, and promotion of p73 activitythrough up-regulation of the interacting partners HSP90 and14-3-3ε. TAp73α expression also promotes the activation oftranscription (RNA pol II), mRNA splicing (serine/arginine-richsplicing factor 1 isoform 1), and translation (eIF-3 complex).Evidence about TAp73α-provoked ER stress (previously

documented with the involvement of Scotin11) were herebyindirectly supported by alterations to protein folding (PDIA5)and might involve RAD23 protein, which takes part in DNA

Figure 5.Results frommultiple reaction monitoring (MRM) analyses ofa short list of selected lipids. Graphpad results are plotted as fold changevariation in TAp73α-induced SAOS-2 cells normalized to noninducedcontrols. Asterisks indicate statistical significance (p-value < 0.05 T testof induced vs noninduced control values and fold-change ≥|2|).

Figure 6.Results frommultiple reaction monitoring (MRM) analyses ofthe main products of lipid peroxidation from untargeted metabolomics.Graphpad results are plotted as fold change variation in TAp73α-induced SAOS-2 cells normalized to noninduced controls. Asterisksindicate statistical significance (p-value < 0.05 T test of induced vsnoninduced control values and fold-change ≥|2|).

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damage repair and ER stress. ER stress was indirectly related atthe metabolic level to ER (and mitochondrial) Ca2+ release,which is known to occur in response to IP3 accumulation(hereby observed upon expression of TAp73α). Moreover, shiftof glycolysis toward PEP accumulation and serine biosynthesis isconsistent with the activation of autophagic signaling.107

Despite sharing several features with other components of thep53 family in terms of downstream effectors, TAp73α favoredpurine metabolism through the up-regulation of ADA synthesisand accumulation of purine metabolites. This results in survivalsignaling, analogously to what has been recently reported for p53in the absence of external stimuli.34

At the metabolic level, TAp73α-dependent induction ofoxidative stress is documented by several indicators, includingthe accumulation of lactoyl-glutathione, the alteration of GSHhomeostasis (related to induction of GLS2 expression), and lipidperoxidation. In this view, up-modulation of the arginine−citrulline−NO metabolism might end up producing NO andpromoting low RNS stress, which in turn would trigger ER stressor high RNS, thereby favoring DNA damage and apoptosis. PPPoveractivation (as gleaned from the TAp73α-dependent up-regulation of 6-phosphogluconolactonase) promoted NADPHproduction as a reducing coenzyme for antioxidant cascades butalso for lipid biosynthesis.Expression of TAp73α in SAOS-2 cells was also accompanied

by increased rates of Krebs metabolism (citrate/isocitrate,succinyl-coA, succinate, malate, fumarate, and FADH2 weredetected in TAp73α-induced SAOS-2 cells) and Krebs-relatedproteins (ATP synthase subunit beta, mitochondrial precursor,and prohibitin), which is consistent with previous reports aboutTAp73α mediating ROS production through mitochondrialactivation and mitochondrial intrinsic apoptotic pathways. Inparticular, the protein prohibitin is also tied to an imbalance inthe synthesis of mitochondrial- and nuclear-encoded mitochon-drial proteins, further supported by TAp73α-mediated down-regulation of mitochondrial ribosomal protein L12.TAp73α also interfered with the fine-tuning of lipid synthesis

and oxidative metabolism, which is supported by either TAp73α-dependent up-regulation of FASN in the literature and hereby bythe lower abundance of enoyl-CoA hydratase in TAp73α-induced in relation to noninduced cells. Lipid metabolism washereby driven toward the accumulation and oxidation of thelong-chain fatty acids with pro-apoptotic potential (polyunsatu-rated 7,10,13,16-docosatetraenoic acid and ceramide 22).Cyclic AMP decreased upon expression of TAp73α, which

should in turn reduce the activation of PKA, whose kinase activityinhibits p73.Regarding the phosphoproteomics analysis, we found

dephosphorylation of several proteins (proteasome subunitalpha type-3, MAP4, GOK, EIF1D, MCM2) associated withspindle checkpoint and mitosis in noninduced cells. It is worthnoting that phosphorylation of these proteins is regulated by thePTEN pathway,108,109 suggesting a potential role for TAp73α inthe regulation of this pathway. PTEN contribution in TAp73α-mediated cascades should result in the inhibition of PI3K, herebyindirectly assessed through the increase in the levels of PIP2 anddecrease of PIP3, which is known to result in the inhibition of Aktand promotion of apoptosis in SAOS-2 cells.In conclusion, the simultaneous application of multiple omics

approaches helped us to draw a global picture of the TAp73αdownstream activated pathways, which paves the way to furtherour understanding the role of p73 either in mediating cell survivalor promoting tumor suppression signaling.

■ ASSOCIATED CONTENT*S Supporting Information

Extended materials and methods, Mascot search results,supplementary table of proteomics data, whole metabolomenetwork in TAp73α-induced (1) and noninduced (2) SAOS-2cells, as gleaned from untargeted metabolomics analyses,extracted ion chromatogram glutathione disulfide (GSSG)from untargeted metabolomics analyses of TAp73α-induced vsnoninduced SAOS-2 cells, pentose phosphate pathway variationin TAp73α-induced SAOS-2 cells normalized against non-induced controls, fatty acid elongation pathway (Kegg 00062)variation in TAp73α-induced SAOS-2 cells normalized againstnoninduced controls, biosynmthesis of unsaturated fatty acidspathway (Kegg 01040) variation in TAp73α-induced SAOS-2cells normalized against noninduced controls, extracted ionchromatogram 7,10,13,16-docosatetraenoic acid from untar-geted metabolomics analyses of TAp73α-induced vs noninducedSAOS-2 cells, and an overview of the pathways downstream ofTAp73α, as resulted from the present integrated omicsinvestigation. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author

*Tel: +39 0761 357 100. Fax: +39 0761 357 630. E-mail address:zolla@unitus.it.Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSA.D.A., C.M., S.R., A.M.T., and L.Z. are supported by funds fromthe Italian National Blood Centre (Centro Nazionale Sangue(CNS), Istituto Superiore Sanita, Rome, Italy). A.D.A. wassupported by student mobility and postdoctoral funds from theInteruniversity Consortium for Biotechnologies (CIB).

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