TRAP1 Regulates Proliferation, Mitochondrial Function, and ......Cell Cycle and Senescence TRAP1...

Cell Cycle and Senescence

TRAP1 Regulates Proliferation, Mitochondrial Function, andHas Prognostic Significance in NSCLC

Jackeline Agorreta1,3, Jianting Hu3, Dongxia Liu3,6, Domenico Delia7, Helen Turley3, David JP. Ferguson3,Francisco Iborra2,4, María J. Pajares1, Marta Larrayoz1, Isabel Zudaire1, Ruben Pio1, Luis M. Montuenga1,Adrian L. Harris4,5, Kevin Gatter3, and Francesco Pezzella3

AbstractThe TNF receptor-associated protein 1 (TRAP1) is a mitochondrial HSP that has been related to drug resistance

and protection from apoptosis in colorectal and prostate cancer. Here, the effect of TRAP1 ablation on cellproliferation, survival, apoptosis, and mitochondrial function was determined in non–small cell lung cancer(NSCLC). In addition, the prognostic value of TRAP1 was evaluated in patients with NSCLC. These resultsdemonstrate that TRAP1 knockdown reduces cell growth and clonogenic cell survival. Moreover, TRAP1downregulation impairs mitochondrial functions such as ATP production and mitochondrial membrane potentialasmeasured byTMRM(tetramethylrhodaminemethylester) uptake, but it does not affectmitochondrial density ormitochondrial morphology. The effect of TRAP1 silencing on apoptosis, analyzed by flow cytometry andimmunoblot expression (cleaved PARP, caspase-9, and caspase-3) was cell line and context dependent. Finally,the prognostic potential of TRAP1 expression in NSCLC was ascertained via immunohistochemical analysiswhich revealed that high TRAP1 expression was associated with increased risk of disease recurrence (univariateanalysis, P ¼ 0.008; multivariate analysis, HR: 2.554; 95% confidence interval, 1.085–6.012; P ¼ 0.03). Inconclusion, these results demonstrate that TRAP1 impacts the viability of NSCLC cells, and that its expression isprognostic in NSCLC.

Implications: TRAP1 controls NSCLC proliferation, apoptosis, and mitochondrial function, and its status hasprognostic potential in NSCLC. Mol Cancer Res; 12(5); 660–9. �2014 AACR.

IntroductionLung cancer is the leading cause of cancer deathworldwide

(1). Non–small cell lung cancer (NSCLC), the most com-mon type of lung cancer, can be subdivided into two mainhistologic subtypes: adenocarcinoma and squamous cellcarcinoma (SCC), accounting for 50% and 30% of all

NSCLC cases, respectively (2). Despite the development oftargeted therapies in lung cancer, there has been littleimprovement in 5-year survival rates. In this context,improved knowledge of the molecular biology of lungcancer, together with biomarkers that predict tumor devel-opment and prognosis, is needed.TNF receptor-associated protein 1 (TRAP1) is a mito-

chondrial protein that belongs to the Hsp90 family, firstidentified as interacting with the intracellular domain of thetype I TNF receptor (3). Later sequence analysis revealedthat TRAP1 was identical to Hsp75 (4). TRAP1 is mainlylocalized in mitochondria of normal and tumor cells (4, 5)acting as a substrate for the serine/threonine kinase PINK1(6). Other localizations include the cytosol, endoplasmicreticulum, and nucleus (7–9). TRAP1 interacts with severalproteins such as retinoblastoma (10), the ATPase TBP7, acomponent of the 19S proteasome regulatory subunit (11),the Ca2þ-binding protein sorcin localized in the mitochon-dria (7, 12), the mitochondrial protein cyclophilin D (5),and the tumor suppressors EXT1 and EXT2, proteinsinvolved in hereditary multiple exostoses (13). Moreover,TRAP1 has been reported to protect against apoptosis(5, 14, 15) and oxidative stress (15–17). Interestingly, ithas been proposed that TRAP1 may be involved in che-moresistance by blocking drug-induced apoptosis in a vari-ety of tumors such as prostate cancer (18), osteosarcoma

Authors' Affiliations: 1Oncology Division, Center for Applied MedicalResearch (CIMA), University of Navarra, Pamplona; 2Department ofMolec-ular and Cellular Biology, Centro Nacional de Biotecnología, ConsejoSuperior de Investigaciones Científicas, Madrid, Spain; 3Nuffield Depart-ment of Clinical Laboratory Sciences; 4Weatherall Institute of MolecularMedicine, University of Oxford, John Radcliffe Hospital; 5Department ofMedical Oncology, University of Oxford, The Churchill Hospital, Oxford,United Kingdom; 6Department of Rheumatology and Immunology, Shan-dong Provincial Hospital, Shandong University, Jinan, China; and 7Depart-ment of Experimental Oncology, Fondazione IRCCS Istituto NazionaleTumori, Milano, Italy

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Authors: Jackeline Agorreta, Oncology Division, Centerfor Applied Medical Research (CIMA), University of Navarra, Pio XII 55,31008 Pamplona, Spain. Phone: 34-948-194-700; Fax: 34-948-194-714;E-mail: [email protected]; andFrancescoPezzella,NuffieldDepartment ofClinical Laboratory Sciences, University of Oxford, JohnRadcliffe Hospital,Headley Way, OX3 9DU Oxford, United Kingdom. Phone: 441865220497;Fax: 441865220519; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-13-0481

�2014 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 12(5) May 2014660

on June 14, 2021. © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst February 24, 2014; DOI: 10.1158/1541-7786.MCR-13-0481

http://mcr.aacrjournals.org/

(15), and colorectal cancer (19). In addition, TRAP1 hasbeen reported to be upregulated in some tumors (5, 18, 20)and downregulated in others (21). TRAP1 has been pro-posed as a candidate biomarker in ovarian and prostatecancer (18, 22) and inhibition of TRAP1 is being exploredas a novel anticancer target (23). In NSCLC, we havepreviously demonstrated that TRAP1-positive cells havehigh levels of cell proliferation promoting genes (21), andthat in the first hours following hypoxia, in absence ofTRAP1, retinoblastoma fails to inhibit proliferation (24).However, the biologic role of this mitochondrial heat shockprotein (HSP) in NSCLC and its relation with mitochon-drial function has not been evaluated yet.The aim of the present study was to determine the role of

TRAP1 on proliferation, cell survival, apoptosis, and mito-chondrial function in lung cancer cell lines and to evaluatethe prognostic role of TRAP1 in patients with NSCLC. Ourresults demonstrate that TRAP1 downregulation reducescell proliferation and survival, induces apoptosis, and impairsmitochondrial functions such as ATP production and mito-chondrial membrane potential regulation. However,TRAP1 knockdown does not affect mitochondrial densityor mitochondrial morphology. In addition, overexpressionof TRAP1 was associated with shorter recurrence-free sur-vival (RFS) in patients with NSCLC.

Materials and MethodsCellsHuman NSCLC cell lines NCI-A549 and NCI-H1299

were obtained from Clare Hall Laboratories and grown inDulbecco's Modified Eagle Medium supplemented with10% FBS and penicillin-streptomycin at 100 U/mL. Cellcultures were incubated at 37�C in a humidified 5% CO2incubator.

Patient samplesA series of 71 patients with a diagnosis of NSCLC who

underwent surgical resection at Clínica Universidad deNavarra (Navarra, Spain) from 2000 through 2008 wereincluded in this study. Clinicopathologic features of thepatients are listed in Table 1. Tumor specimens wereclassified according to the 2004 World Health Organi-zation criteria (25). The inclusion criteria were NSCLChistology, no neoadjuvant chemo- or radiotherapy, andabsence of cancer within the 5 years previous to lungcancer surgery. The study protocol was approved by theinstitutional medical ethical committee. Writteninformed consent was obtained from each patient beforeparticipation. RFS was calculated from the date of surgeryto the date of detection of recurrence or the date of thelast follow-up. The median follow-up time was 42months.

Immunohistochemistry in clinical specimens frompatients with NSCLCFormalin-fixed paraffin-embedded tissue sections were

evaluated. Endogenous peroxidase activity was quenched

and antigen retrieval was carried out by pressure cookingin 10 mmol/L citrate buffer, pH 6. Nonspecific bindingwas blocked using 5% normal goat serum in Tris-bufferedsaline for 30 minutes. Sections were incubated with anti-TRAP1 antibody (1:400; Labvision) overnight at 4�C.Sections were then incubated with Envision polymer(Dako) for 30 minutes at room temperature. Peroxidaseactivity was developed using diaminobenzidine and coun-terstained with hematoxylin before mounting in DPXmedium (BDH Chemical). The specificity of TRAP1antibody was demonstrated using a variety of controls,including Western blot analysis, inhibition with TRAP1-siRNA sequences, isotype control, and omission of theprimary antibody.

Immunostaining evaluationTwo independent, blinded observers (F. Pezzella and

J. Agorreta) evaluated the intensity and extensiveness ofstaining in all of the study samples. The evaluation ofcytoplasmic TRAP1 expression was performed using theH-score system (26). Briefly, the percentage of positive cells(0%–100%) and the intensity of staining (1þ, mild; 2þ,moderate; and 3þ, intense labeling) were scored. Disagree-ments were resolved by common reevaluation.

ImmunoblottingProtein and total RNA were extracted using Paris kit

(Ambion-Life Technologies Ltd) according to the manu-facturer's instructions. Thirty micrograms of total proteinfrom each lysate were boiled at 95�C for 5minutes, separatedby SDS/PAGE under reduced conditions (5% 2-

Table 1. Clinicopathological characteristics ofthe patients

N ¼ 71Age, y (median–interquartile range) 63 (54–70)

mercaptoethanol), and transferred onto a nitrocellulosemembrane. The membranes were subsequently blocked in5% defattedmilk-PBS for 1 hour and incubated overnight at4�C with a primary antibody anti TRAP1 (1:1000, Labvi-sion) or anti b-actin (1:10000, Sigma). Blots were thenincubated with a horseradish peroxidase-linked secondaryantibody (1:5000; Amersham Pharmacia Biotech) anddeveloped by chemoluminiscence with Lumilight Plus Kit(Roche diagnostics). Apoptosis detection by Western blot-ting was performed as described before (27).

RNA interferenceFor inhibition of TRAP1 expression, cells were seeded

(1 � 106 cells per well) in 10 cm dishes in antibiotic-freemedium. At 24 hours, cells were transfected with 40 nmol/Lof siRNA by using Oligofectamine (Invitrogen-Life Tech-nologies Ltd) as previously described (21). Two siRNAsequences against TRAP1 were designed and synthesized byEurogentec (TRAP1-siRNA1: 50-AUGUUUGGAAGUG-GAACCC-30 and 50-ACCAUCUGAAAGCCACUGG-30;TRAP1-siRNA2: 50-TGCTGTTTGGAAGTGGAACCC-TGCACGTTTTGGCCACTGACTGACGTGCAGGGC-CACTTCCAAA-30 and 50-CCTGTTTGGAAGTGGCC-CTGCACGTCAGTCAGTGGCCAAAACGTGCAGGG-TTCCACTTCCAAAC-30). A scrambled (scr) siRNA (50-AUGUUUGGAAGUGGAACCC-30 and 50-UAGGGU-GUACCCGUAAUAG-30) was used as the negative control.

RT-PCRRetrotranscription was performed using RetroScript Kit

(Ambion). TRAP1 and b-actin expression was analyzed byPCR using TaqMan Gene Expression Assays (AppliedBiosystems). The reaction was performed on a PTC-200thermal cycler with a Chromo 4 continuous fluorescencedetector (Bio-Rad). The comparative cycle threshold (Ct)method was used to analyze the data by generating relativevalues of the amount of target cDNA, according to the2�DDCt method (28) using b-actin as endogenous gene andscramble (scr) expression as calibrator.

Growth curvesCells were seeded on 6-well dishes at a density of 1� 105

cells per well in triplicate and cultured for 1 to 7 days.Subsequently, cell number was assessed with a Coulter Z2particle count and size analyzer (Beckman Coulter). Auto-matically cell count was carried out with a Cell IQ micro-scope (Chipman Technologies).

Clonogenic assayTwenty four hours after siRNA transfection, cells were

harvested, seeded in triplicate (300 cells per well) in 6-wellplates, and incubated at 37�C in a 5% CO2 atmosphere.After 14 days, colonies were fixed in methanol-acetic acid(1:1), stained with crystal violet, and counted.

Proliferation index determinationsiRNA-treated cells were seeded in 10 cm dishes and

grown for 1, 3, or 5 days. Subsequently, cells were

harvested and fixed overnight in 4% phosphate-bufferedformalin (pH 7.0), suspended in agar, and embedded inparaffin. Antigen retrieval was carried out in 3 mm sectionsby pressure cooking in 10 mmol/L citrate buffer, pH 6,and immunohistochemical staining for the human Ki-67protein was performed using the anti-MIB1 antigen anti-body (Dako) at 1:50 for 30 minutes at room temperature.Sections were incubated with the Envision detectionsystem (Dako) and developed with diaminobenzidine.Immunohistochemical scoring was performed as previous-ly described (29).

Cell-cycle and apoptosis analysisCell-cycle analyses were performed on trypsin-disaggre-

gated cryopreserved cell suspensions containing floating andattached cells. Following thawing, cells were centrifuged toremove the cryopreservation solution (10% dimethyl sulf-oxide in FBS), fixed in 70% ethanol on ice, treated with 1mg/mL RNase, stained with 10 mg/mL propidium iodide,and examined with a FACSCalibur instrument fitted with aCell Quest software package (BD Biosciences). About50,000 cells per sample were analyzed. Percentages of cellsin the Sub-G1, G1, S and G2–M phases were determined.For apoptosis analysis, fresh trypsin-disaggregated cell sus-pensions containing floating and attached cells were used aspreviously described (30). Briefly, cells were washed andstained with 2 mL of Annexin V (BD Biosciences) and 2 mLof 10 mg/mL of propidium iodide (Sigma). Apoptosis wasinduced by staurosporine treatment (1 mg/mL for 4 h).Samples were analyzed on a FACSCalibur instrument andquadrant analysis was performed with FlowJo 9.3 software(Tree Star). At least three independent experiments percondition were performed.

Mitochondrial functionThe amount of ATP was measured in lysates of 105 cells

using the ATP Bioluminescence Assay Kit (Roche) inaccordance with themanufacturer's instructions. Thismeth-od uses the ATP dependency of the light-emitting, lucifer-ase-catalyzed oxidation of luciferin for the measurement ofATP concentration. To analyze the mitochondrial mem-brane potential, TMRM (tetramethylrhodamine methyles-ter; Invitrogen) staining was used because it is a cell-per-meant, cationic, red-orange fluorescent dye that is readilysequestered by active mitochondria. MitoTracker Greenstaining (Molecular Probes-Life Technologies Ltd) was alsoused tomeasure mitochondrial mass regardless of mitochon-drial membrane potential. Moreover, the production ofreactive oxygen species (ROS) was evaluated by the Mito-SOX staining (Molecular probes) as previously described(31). Fluorescence images were collected using a confocalmicroscope (Zeiss LSM 510 META; Carl Zeiss) and fluo-rescence intensity wasmeasuredwith ImageJ software (NIH,Bethesda, MD).

Electron microscopyCells were fixed in 4% glutaraldehyde in 0.1 mol/L

phosphate buffer and processed for routine electron

Agorreta et al.

Mol Cancer Res; 12(5) May 2014 Molecular Cancer Research662




microscopy as previously described (32). Mitochondrialmass was measured with ImageJ software.

Statistical analysisStatistical analysis was performed using SPSS 15.0. Data

obtained from cell count, colony formation, MIB1 stain-ing, cell cycle, and mitochondrial function experimentswere analyzed by the Student t test or the Mann–WhitneyU test for parametric and nonparametric variables, respec-tively. For survival analysis, Kaplan–Meier survival curvesand the log-rank test were used to analyze differences inRFS (the median was selected as the cutoff value). Mul-tivariate analysis was carried out using the Cox propor-tional hazards model. Only variables of P < 0.1 from theunivariate analysis were entered in the Cox regressionanalysis. The proportional hazards assumption was

examined by testing interactions between the covariatesof the final model and time. P < 0.05 was consideredstatistically significant.

ResultsExpression of TRAP1 is necessary for cell growthTo examine the effect of TRAP1 inhibition on cell

proliferation, we carried out downregulation experimentsin lung cancer cell lines. Knockdown was carried out inH1299 and A549 cells using two different siRNAs and theefficacy of TRAP1 siRNA downregulation was verified byWestern blotting and real-time PCR (RT-PCR; Fig. 1A andB). TRAP1 downregulation resulted in a significant reduc-tion in cell growth in both H1299 and A549 cell lines asconfirmed by TRAP1-siRNA1 and 2 sequences (Fig. 1C).Cell growth of TRAP1-siRNA1–treated A549 cells was also

Figure 1. TRAP1 knockdown inhibits cell proliferation and survival on the H1299 and A549 cell lines. Successful knockdown of TRAP1 expressionby two independent TRAP1-siRNA sequences was demonstrated by RT-PCR (A) and Western blot analysis (B) in both H1299 and A549 celllines at day 4. To determine the effect of TRAP1 siRNA knockdown on tumor cell proliferation, cells were transfected with control- (scr) orTRAP1-siRNAs and cell number was determined by a Coulter Z2 particle count and size analyzer (C) or automatically determined by a Cell IQmicroscope (D). E, TRAP1 downregulation significantly reduced colony formation in the A549 and H1299 cell lines. Data, mean � SD from at least threeindependent experiments.

TRAP1 Regulates NSCLC Proliferation and Mitochondrial Function

www.aacrjournals.org Mol Cancer Res; 12(5) May 2014 663




monitored by time-lapse video microscopy for 5 days at a35- minute interval, confirming the reduction in cellnumber after TRAP1 knockdown (Fig. 1D and Supplemen-tary Movies S1 and S2). The impairment of cell survivalwas further confirmed by clonogenic assay in H1299 andA549 cell lines (Fig. 1E). We next investigated the effect ofTRAP1 knockdown on cell proliferation by staining cellpellets of scr- and TRAP1-siRNA A549-treated cells atdifferent time points for ki-67 protein (MIB1 antigen).We found that from day 3, there was a significant reductionofMIB1-positive cells when TRAP1was inhibited (Fig. 2A).Cell-cycle analysis by flow cytometry showed a significantreduction in the percentage of cells in G2–M phase afterTRAP1 knockdown (Fig. 2B and C), confirming theresults from the immunohistochemical analysis of ki-67expression.

TRAP1 downregulation has a variable effect onapoptosisQuantification of apoptotic cells by Annexin V/PI assay

showed that TRAP1 downregulated A549 cells hadincreased apoptotic rates as compared with scr-siRNAtreated (Fig. 2D, top). Those effects were less evident inH1299 cell line (Supplementary Fig. S1). The inductionof apoptosis in A549 cells was confirmed by the increaseof activated (cleaved) caspase-3, caspase-9, and PARP(Fig. 2E, left). It should be noted that when apoptosiswas induced by treating cells with staurosporine, there wasa dramatic induction of apoptosis in TRAP1-siRNA–treated cells but not in scr-control cells (Fig. 2D, bottomand Fig. 2E, right). The cell line H1299 did not showclear evidence of apoptosis as silencing of TRAP produceda mild increase of PARP but a similarly mild decrease of

Figure 2. Downregulation of TRAP1 arrests cell proliferation and induces apoptosis in the A549 lung cancer cell line. A, proliferative fraction given by thepercentage of ki-67–positive cells was significantly reduced in TRAP1-siRNA1–treated cells. B, cell-cycle distribution of A549 cells at different daysafter TRAP1-siRNA1 transfection. C, differences in the percentage of cells in S and G2–M phases at day 3. D, Annexin V/propidium iodide staining wasperformed on A549 cells transfected with scr, TRAP1-siRNA1, and TRAP1-siRNA2 sequences (top) or transfected cells treated with 1 mg/mLstaurosporine (bottom) and analyzed by flow cytometry. Percentages of intact cells (Annexin V� PI�), early apoptotic cells (Annexin Vþ PI�) and lateapoptotic or necrotic cells (Annexin Vþ PIþ) are shown in the plot. One representative experiment is shown from three independent repetitions. E,apoptosis was also demonstrated by Western blot analysis of cleaved PARP and cleaved caspase-3 and -9 in A549 cells. Data, mean� SD from at leastthree independent experiments.

Agorreta et al.





cleaved caspase-9 while there was no evidence of caspase-3activation (Supplementary Fig. S1).No morphologic evidence of accumulation of apoptotic

bodies could be seen in the cell culture movies (Supplemen-tary Movies).

TRAP1 downregulation impairs mitochondrial functionWe hypothesized that the effects of TRAP1 inhibition

could be caused by mitochondrial dysfunction becauseTRAP1 is known to be mainly expressed in the mitochon-dria. Therefore, we analyzed a variety of mitochondrialfunctions after TRAP1 inhibition. First, we analyzed ATPproduction in scr- and TRAP1-siRNA1–treated cells, show-ing that TRAP1 inhibition was associated with a 30%reduction of ATP (P¼ 0.002; Fig. 3A). Next, we examinedthe effect of TRAP1 expression onmitochondrial membranepotential as measured by TMRM uptake. A significant

reduction on membrane potential was shown in TRAP1-siRNA1–treated cells as compared with scr-siRNA (P <0.001; Fig. 3B and C). However, no differences were foundin the mitochondrial mass measured by MitoTracker stain-ing (P¼ 0.809; Fig. 3D) or in ROS productionmeasured byMitoSOX staining (P ¼ 0.078; Fig. 3E). Electron micros-copy also did not demonstrate changes in mitochondrialmorphology or mitochondrial mass (Fig. 3F).

High TRAP1 expression is associated with worseprognosis in patients with NSCLCTRAP1 expression was analyzed by immunohistochem-

istry in a series of 71 patients withNSCLC.Themain clinicaland pathologic characteristics of these patients are summa-rized in Table 1. Moderate TRAP1 staining was found innormal bronchial mucosa adjacent to the tumor (Fig. 4A),although no immunoreactivity was found in alveoli (Fig.

Figure 3. TRAP1 downregulation impairs mitochondrial function in A549 cells. A, measurement of ATP levels by a bioluminescence assay demonstratesthat TRAP1 inhibition reduces ATP levels. B, representative images of TMRM uptake in scr- and TRAP1-siRNA1–treated cells. C, quantification of TMRMuptake by image analysis shows a reduction in mitochondrial membrane potential in TRAP1-siRNA1–treated A549 cells as compared with controlcells. D, mitochondrial mass measured by MitoTracker staining was similar in scr and TRAP1-siRNA1–treated cells. E, no differences in ROS production,as determined by MitoSOX staining, were found. F, the ultrastructure of the mitochondria was visualized using transmission electron microscopy, and nodifferences in the mitochondrial morphology were observed between scr- and TRAP1-siRNA1–treated cells. Moreover, no significant differences inmitochondrial mass were found. Scale bar, 500 nm. Data, mean � SD from three independent experiments.






4B). In tumor samples, TRAP1 staining was observedpredominantly in the cytoplasm of all tumors analyzed (Fig.4C). In some cases, TRAP1 staining was both in the nucleusand in the cytoplasm (Fig. 4D).We next sought to evaluate the prognostic role of TRAP1

expression in our cohort of NSCLC. Nuclear expression ofTRAP1 did not correlate with prognosis (log-rank test; P ¼0.486). However, when cytoplasmic TRAP1 expression wasanalyzed, those patients with high TRAP1 expression (usingthe median as the cutoff value) showed shorter RFS thanpatients with low levels of TRAP1 (P ¼ 0.008; Fig. 4E).Multivariate analysis using Cox regression model was per-formed to determine the independent prognostic factors.Relevant clinicopathological variables such as age, gender,stage, histology, and smoking history, as well as cytoplasmicTRAP1 expression, were analyzed in the Cox univariatemodel, and only those variables with a P value < 0.1 (stageand TRAP1 expression) were included in the multivariate

model. The Cox regression analysis revealed that highcytoplasmic TRAP1 expression was an independent predic-tor of shorter RFS when patients were adjusted by stage [HR¼ 2.554; confidence interval (CI), 1.085–6.012; Table 2].

100

80

60

40

20

00 20 40 60 80 100 120

Follow-up time (months)

% R

ecu

rren

ce-f

ree

surv

ival

High expression

Low expression

P = 0.008

A B

C D

E

Figure 4. High cytoplasmic TRAP1staining is associated withadverse prognosis in NSCLC.Representative TRAP1immunostaining in bronchialepithelium (A), lung parenchyma(B), lung adenocarcinoma (C), andSCC of the lung (D). E, Kaplan–Meier RFS curves for TRAP1expression and log-rank test.Shorter RFS time was found intumors bearing high TRAP1expression. Scale bar, 50 mm.

Table 2. Multivariate Cox regression analysis ofRFS in patients with NSCLC

HR (95% CI) P

Cytoplasmic TRAP1 expressionLow — —High 2.554 (1.085–6.012) P ¼ 0.032

StageI, II — —III, IV 1.720 (0.725–4.077) P ¼ 0.218

Agorreta et al.





These results demonstrate that TRAP1 expression correlateswith poor outcome in patients with NSCLC.

DiscussionIn the present study, we have demonstrated that TRAP1

has important effects on mitochondrial function and plays akey role in the regulation of proliferation, survival, andapoptosis of NSCLC cells. Moreover, we have shown thathigh cytoplasmic TRAP1 expression is associated with worseprognosis in patients with NSCLC.During the last 10 years, an increasing number of studies

have demonstrated the versatility of TRAP1 protein and itsinvolvement in a number of pathways. Originally clonedbecause of its interaction with TNF receptor, and thereforelikely to be involved in cell signaling (3, 4), it was then foundthat TRAP1 also acts as a chaperon to retinoblastoma,maintaining retinoblastoma protein in its active conforma-tion (10). The importance of the role of TRAP1 as chaperonhas quickly outgrown its original role in retinoblastomaas its pivotal role in cytoprotection has emerged. TRAP1has been described to protect mitochondria from oxidativestress and ROS (reviewed in refs. 33–35). In this sense,TRAP1 blocksROS activity (15), ROSproduction (36), andregulates the mitochondrial permeability transition pores(5, 37). It has been recently demonstrated that TRAP1 hasan important role controlling central metabolic networksin the mitochondria of tumor cells (38, 39). All thesefunctions are believed to be important for its role in protect-ing from apoptosis and inducing chemoresistance (11, 33).In the present study, we investigated the role of TRAP1 inNSCLC cell lines growth by looking at its effects on cellproliferation, apoptosis, and mitochondrial function.Downregulation of TRAP1 expression in NSCLC cell

lines produced a significant reduction of cell proliferationand survival as assessed by cell count, clonogenic assays, ki-67 expression, and cell-cycle analysis. In agreement withthese results, proliferation had been previously linked toTRAP1 by our group (24) and others (40). However, there isno general agreement in literature as some authors have failedto notice any effect on cell growth (38). Moreover, we havedetermined that TRAP1 knockdown is associated with asubtle and variable effect on induction of apoptosis. Thisinduction becomes more evident when apoptosis is inducedpharmacologically with staurosporine. Our results havedemonstrated a variable role in antiapoptotic functions ofTRAP1 in NSCLC cell lines with clear signs of apoptosisdetected in one of the cell lines studied. These results are stillin agreement with previous reports showing a protectiveeffect of TRAP1 against apoptosis-inducing anticancer drugsor ROS (12, 14, 15, 18, 19). Having taken all these data intoaccount, we suggest that in many types of cells, loss ofTRAP1 has a mitochondrial priming effect which tips thecells toward apoptosis making them more sensitive to theeffects of cytotoxic drugs (41, 42). The observation thatapoptosis is obvious in one of the two cell lines analyzed,independently from the pharmacologic stimulation, furthersupport the protective role of TRAP1 on apoptosis as itsdownregulation can be the last step of the priming in some

lines (e.g., A549) but an intermediate in others (e.g.,H1299).Moreover, we have demonstrated herein that TRAP1

inhibition leads to a reduction of ATP production andmitochondrial membrane potential. These findings are con-sistent with previous results indicating that TRAP1 in cancerprevents mitochondrial damage (33) and after its inhibition,the misfolding of Peptidylprolyl isomerases D results inloss of ATP production (5) and are also consistent with itsprotective role from apoptosis. The role of TRAP1 inmaintaining ATP levels has also been reported in rat brain(43). In contrast, two recent papers (40, 44) report that lossof TRAP1 results in an increase of ATP production follow-ing a switch from glycolysis to oxidative phosphorylation.We cannot provide an explanation for this discrepancy asfar as the role of TRAP1 in ATP production is concerned,but it is likely to be with the different types of final effectsthat TRAP1 can have according to the type of cell beinginvestigated.We have previously demonstrated that after a short

hypoxic shock, TRAP1 translocates to the nucleus and isessential to maintain retinoblastoma suppressor gene func-tion: in its absence, the cells fail to slow down proliferation(24). However, this is a short-term effect and, in agreementwith our previous results and as demonstrated here, innormoxia and in the longer term TRAP1 promotes the cellcycle. Therefore, it seems to have two opposite functions: innormoxia, TRAP1 promotes cell proliferation and protectsfrom apoptosis; however, following a hypoxic shock itmovesto the nucleus where it is essential for retinoblastoma toinduce a rapid, short-term slowing down of proliferation. Ifhypoxia persists, TRAP1 cytoplasmic levels will decrease(unpublished results) alongside a diminution of the prolif-eration rate. In this respect, it can be considered to haveoncogenic properties as suggested by Sciacovelli and collea-gues (40) although the potential suppressor effect in malig-nancies, due to its chaperone role to retinoblastoma (10),remains to be elucidated.On the other hand, TRAP1 expression has also been

correlated with chemoresistance in breast, colorectal, andovarian carcinoma (7, 19, 43, 45). Although there is con-sensus that TRAP1 expression is a predictive biomarker fordrug resistance, because of the broad range of cellular func-tions influenced by TRAP1 (33), it is perhaps not surprisingthat its role as immunohistochemical prognostic biomarkeris controversial. As a matter of fact, a role as oncogene (40),together with a role as tumor suppressor gene (44), has beenproposed. Our data obtained from lung primary tumorsshowed both nuclear and cytoplasmic staining of TRAP1 intumor cells, as it was previously reported in NSCLC, breastcarcinoma, ovarian cancer, and other malignancies (18,24, 45). Only a few studies have been performed looking atTRAP1 expression on tumor samples and correlations withprognosis (24, 45, 46), and the results are controversial. Incolorectal carcinoma, high TRAP1 expression has been cor-related with shorter RFS and overall survival (OS; ref. 46).However, in a study on ovarian carcinoma, cytoplasmicexpression of TRAP1 was associated with better OS, whereas






no association was found with RFS (45). Finally, in a series ofbreast carcinoma, we found no correlation between cyto-plasmic TRAP1 and OS or RFS, although nuclear TRAP1expression was instead associated with both retinoblastomapositivity and longer RFS but no association was found withOS (24). In the present study, we demonstrated that cyto-plasmic expression of TRAP1 is an independent predictor ofthe shortest relapse-free survival in surgically resectedNSCLC. To our knowledge, this is the first study thatanalyzes the role of TRAP1 in the prognosis of lung cancer.Although the causes underlying diverse results in differenttumor types are still unknown, we may argue that TRAP1plays organ-specific roles in each tumor type. Indeed, itshould be noted that the association between cytoplasmicstaining and worse prognosis demonstrated in NSCLC inthe present study and in colorectal carcinoma (46) is fullyconsistent with the suggested role of TRAP1 in cell prolif-eration and protection to apoptosis.However,more extensivestudies exploring the role of TRAP1 as a potential target orpredictor of response in NSCLC are warranted.In conclusion, the complexity of the role played by

TRAP1 in the cancer cell biology is the most likely expla-nation for the discrepant results observed in literature;however, our data further support the role played by TRAP1in mitochondrial function and regulation of apoptosis,but also demonstrate a role in cell growth through theregulation of the cell cycle. Furthermore, we have demon-strated that high TRAP1 expression is an adverse prognosticfactor for patients with NSCLC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Agorreta, J. Hu, A.L. Harris, K. Gatter, F. PezzellaDevelopment of methodology: J. Agorreta, J. Hu, H. Turley, F. Iborra, F. PezzellaAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): J. Agorreta, J. Hu, D. Delia, D.J.P. Ferguson, F. Iborra, M.J. Pajares,L.M. Montuenga, A.L. Harris, F. PezzellaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Agorreta, J. Hu, D.J.P. Ferguson, I. Zudaire, R. Pio,L.M. Montuenga, A.L. Harris, F. PezzellaWriting, review, and/or revision of the manuscript: J. Agorreta, J. Hu, D. Liu,D. Delia, H. Turley, M.J. Pajares, M. Larrayoz, I. Zudaire, R. Pio, L.M. Montuenga,A.L. Harris, K. Gatter, F. PezzellaAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): J. Agorreta, M. Larrayoz, K. Gatter, F. PezzellaStudy supervision: J. Agorreta, J. Hu, A.L. Harris, F. Pezzella

AcknowledgmentsThe authors thank G. Steers, Drs. R. Leek, K. Giaslakiotis, H. Mellor and

S. Wigfield for technical support.

Grant SupportThis work was supported by Cancer Research UK grants and "UTE project

CIMA". J. Agorreta was funded by the Sara Borrell Program of ISCIII, SpanishGovernment.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 10, 2013; revised January 27, 2014; accepted January 30,2014; published OnlineFirst February 24, 2014.

References1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer

J Clin 2013;63:11–30.2. Perez-Moreno P, Brambilla E, Thomas R, Soria JC. Squamous cell

carcinoma of the lung: molecular subtypes and therapeutic opportu-nities. Clin Cancer Res 2012;18:2443–51.

3. Song HY, Dunbar JD, Zhang YX, Guo D, Donner DB. Identification of aprotein with homology to hsp90 that binds the type 1 tumor necrosisfactor receptor. J Biol Chem 1995;270:3574–81.

4. Felts SJ, Owen BA, Nguyen P, Trepel J, Donner DB, Toft DO. Thehsp90-related protein TRAP1 is a mitochondrial protein with distinctfunctional properties. J Biol Chem 2000;275:3305–12.

5. Kang BH, Plescia J, Dohi T, Rosa J, Doxsey SJ, Altieri DC. Regulationof tumor cell mitochondrial homeostasis by an organelle-specificHsp90 chaperone network. Cell 2007;131:257–70.

6. Pridgeon JW, Olzmann JA, Chin LS, Li L. PINK1 protects againstoxidative stress by phosphorylating mitochondrial chaperone TRAP1.PLoS Biol 2007;5:e172.

7. Maddalena F, Sisinni L, Lettini G, Condelli V, Matassa DS, Piscazzi A,et al. Resistance to paclitxel in breast carcinoma cells requires a qualitycontrol of mitochondrial antiapoptotic proteins by TRAP1. Mol Oncol2013;7:895–906.

8. Cechetto JD, Gupta RS. Immunoelectron microscopy provides evi-dence that tumor necrosis factor receptor-associatedprotein 1 (TRAP-1) is a mitochondrial protein which also localizes at specific extra-mitochondrial sites. Exp Cell Res 2000;260:30–9.

9. Takemoto K, Miyata S, Takamura H, Katayama T, Tohyama M. Mito-chondrial TRAP1 regulates the unfolded protein response in theendoplasmic reticulum. Neurochem Int 2011;58:880–7.

10. ChenCF, Chen Y, Dai K, Chen PL, Riley DJ, LeeWH. A newmember ofthe hsp90 family of molecular chaperones interacts with the retino-

blastoma protein during mitosis and after heat shock. Mol Cell Biol1996;16:4691–9.

11. Amoroso MR, Matassa DS, Laudiero G, Egorova AV, Polishchuk RS,Maddalena F, et al. TRAP1 and the proteasome regulatory particleTBP7/Rpt3 interact in the endoplasmic reticulum and control cellularubiquitination of specific mitochondrial proteins. Cell Death Differ2012;19:592–604.

12. Landriscina M, Laudiero G, Maddalena F, Amoroso MR, Piscazzi A,Cozzolino F, et al. Mitochondrial chaperone Trap1 and the calciumbinding protein Sorcin interact and protect cells against apoptosisinduced by antiblastic agents. Cancer Res 2010;70:6577–86.

13. Simmons AD, Musy MM, Lopes CS, Hwang LY, Yang YP, Lovett M. Adirect interaction between EXT proteins and glycosyltransferases isdefective in hereditary multiple exostoses. Hum Mol Genet 1999;8:2155–64.

14. Masuda Y, Shima G, Aiuchi T, Horie M, Hori K, Nakajo S, et al.Involvement of tumor necrosis factor receptor-associated protein 1(TRAP1) in apoptosis induced by beta-hydroxyisovalerylshikonin.J Biol Chem 2004;279:42503–15.

15. MontesanoGesualdiN,ChiricoG,PirozziG,CostantinoE,LandriscinaM,Esposito F. Tumor necrosis factor-associated protein 1 (TRAP-1) pro-tects cells from oxidative stress and apoptosis. Stress 2007;10:342–50.

16. Im CN, Lee JS, Zheng Y, Seo JS. Iron chelation study in a normalhuman hepatocyte cell line suggests that tumor necrosis factor recep-tor-associated protein 1 (TRAP1) regulates production of reactiveoxygen species. J Cell Biochem 2007;100:474–86.

17. Voloboueva LA, Duan M, Ouyang Y, Emery JF, Stoy C, Giffard RG.Overexpression of mitochondrial Hsp70/Hsp75 protects astrocytesagainst ischemic injury in vitro. J Cereb Blood Flow Metab 2008;28:1009–16.

Agorreta et al.





18. Leav I, Plescia J, Goel HL, Li J, Jiang Z, Cohen RJ, et al. Cytoprotectivemitochondrial chaperone TRAP-1 as a novel molecular target inlocalized and metastatic prostate cancer. Am J Pathol 2010;176:393–401.

19. Costantino E, Maddalena F, Calise S, Piscazzi A, Tirino V, Fersini A,et al. TRAP1, a novel mitochondrial chaperone responsible for multi-drug resistance and protection from apoptotis in human colorectalcarcinoma cells. Cancer Lett 2009;279:39–46.

20. Fang W, Li X, Jiang Q, Liu Z, Yang H, Wang S, et al. Transcriptionalpatterns, biomarkers and pathways characterizing nasopharyngealcarcinoma of Southern China. J Transl Med 2008;6:32.

21. Liu D, Hu J, Agorreta J, Cesario A, Zhang Y, Harris AL, et al. Tumornecrosis factor receptor-associated protein 1(TRAP1) regulates genesinvolved in cell cycle andmetastases. Cancer Lett 2010;296:194–205.

22. Landriscina M, Amoroso MR, Piscazzi A, Esposito F. Heat shockproteins, cell survival and drug resistance: the mitochondrial chaper-one TRAP1, a potential novel target for ovarian cancer therapy.Gynecol Oncol 2010;117:177–82.

23. Siegelin MD. Inhibition of the mitochondrial Hsp90 chaperonenetwork: a novel, efficient treatment strategy for cancer? CancerLett 2013;333:133–46.

24. Hu J, Tan EY, Campo L, Leek R, Seman Z, Turley H, et al. TRAP1 isinvolved in cell cycle regulated by retinoblastoma susceptibility gene(RB1) in early hypoxia and has variable expression patterns in humantumors. Journal of Cancer Research Updates 2013;2:194–210.

25. Travis WD, Brambilla E, M€uller-Hermelink HK, Harris CC. Tumoursof the Lung, Pleura, Thymus and Heart. Lyon, France: IARC Press;2004.

26. PajaresMJ,Agorreta J, LarrayozM,VesinA, Ezponda T, Zudaire I, et al.Expression of tumor-derived vascular endothelial growth factor and itsreceptors is associated with outcome in early squamous cell carcino-ma of the lung. J Clin Oncol 2012;30:1129–36.

27. Cossu F, Milani M, Vachette P, Malvezzi F, Grassi S, Lecis D, et al.Structural insight into inhibitor of apoptosis proteins recognition by apotent divalent smac-mimetic. PLoS ONE 2012;7:e49527.

28. Livak KJ, Schmittgen TD. Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods 2001;25:402–8.

29. Sington J,Giatromanolaki A, Campo L, TurleyH, Pezzella F,Gatter KC.BNIP3 expression in follicular lymphoma. Histopathology 2007;50:555–60.

30. deMiguel FJ, Sharma RD, PajaresMJ,Montuenga LM, Rubio A, Pio R.Identification of alternative splicing events regulated by the oncogenicfactor SRSF1 in lung cancer. Cancer Res 2014;74:1105–15.

31. das Neves RP, Jones NS, Andreu L, Gupta R, Enver T, Iborra FJ.Connecting variability in global transcription rate to mitochondrialvariability. PLoS Biol 2010;8:e1000560.

32. Sivridis E, Koukourakis MI, Zois CE, Ledaki I, Ferguson DJ, Harris AL,et al. LC3A-positive light microscopy detected patterns of autophagyand prognosis in operable breast carcinomas. Am J Pathol 2010;176:2477–89.

33. Altieri DC, Stein GS, Lian JB, Languino LR. TRAP-1, the mitochondrialHsp90. Biochim Biophys Acta 2012;1823:767–73.

34. Kang BH. TRAP1 regulation of mitochondrial life or death decision incancer cells and mitochondria-targeted TRAP1 inhibitors. BMB Rep2012;45:1–6.

35. Matassa DS, Amoroso MR, Maddalena F, Landriscina M, Esposito F.New insights into TRAP1 pathway. Am J Cancer Res 2012;2:235–48.

36. Hua G, Zhang Q, Fan Z. Heat shock protein 75 (TRAP1) antagonizesreactive oxygen species generation and protects cells from granzymeM-mediated apoptosis. J Biol Chem 2007;282:20553–60.

37. Xiang F, HuangYS, Shi XH, ZhangQ.Mitochondrial chaperone tumournecrosis factor receptor-associated protein 1 protects cardiomyo-cytes from hypoxic injury by regulating mitochondrial permeabilitytransition pore opening. FEBS J 2010;277:1929–38.

38. Caino MC, Chae YC, Vaira V, Ferrero S, Nosotti M, Martin NM, et al.Metabolic stress regulates cytoskeletal dynamics and metastasis ofcancer cells. J Clin Invest 2013;123:2907–20.

39. Chae YC, Angelin A, Lisanti S, Kossenkov AV, Speicher KD, Wang H,et al. Landscape of the mitochondrial Hsp90 metabolome in tumours.Nat Commun 2013;4:2139.

40. Sciacovelli M, Guzzo G, Morello V, Frezza C, Zheng L, Nannini N, et al.The mitochondrial chaperone TRAP1 promotes neoplastic growth byinhibiting succinate dehydrogenase. Cell Metab 2013;17:988–99.

41. Ni Chonghaile T, Sarosiek KA, Vo TT, Ryan JA, Tammareddi A, MooreVdel G, et al. Pretreatment mitochondrial priming correlates withclinical response to cytotoxic chemotherapy. Science 2011;334:1129–33.

42. Sarosiek KA, Ni Chonghaile T, Letai A. Mitochondria: gatekeepers ofresponse to chemotherapy. Trends Cell Biol 2013;23:612–9.

43. ChienWL, Lee TR, Hung SY, Kang KH, LeeMJ, FuWM. Impairment ofoxidative stress-induced heme oxygenase-1 expression by the defectof Parkinson-related gene of PINK1. J Neurochem 2011;117:643–53.

44. Yoshida S, Tsutsumi S, Muhlebach G, Sourbier C, LeeMJ, Lee S, et al.Molecular chaperone TRAP1 regulates a metabolic switch betweenmitochondrial respiration and aerobic glycolysis. Proc Natl Acad SciU S A 2013;110:E1604–12.

45. Aust S, Bachmayr-Heyda A, Pateisky P, Tong D, Darb-Esfahani S,Denkert C, et al. Role of TRAP1 and estrogen receptor alpha in patientswith ovarian cancer: A study of the OVCAD consortium. Mol Cancer2012;11:69.

46. Gao JY, Song BR, Peng JJ, Lu YM. Correlation betweenmitochondrialTRAP-1 expression and lymph node metastasis in colorectal cancer.World J Gastroenterol 2012;18:5965–71.






2014;12:660-669. Published OnlineFirst February 24, 2014.Mol Cancer Res Jackeline Agorreta, Jianting Hu, Dongxia Liu, et al. Prognostic Significance in NSCLCTRAP1 Regulates Proliferation, Mitochondrial Function, and Has

Updated version

10.1158/1541-7786.MCR-13-0481doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2014/02/27/1541-7786.MCR-13-0481.DC1

Access the most recent supplemental material at:

Cited articles

http://mcr.aacrjournals.org/content/12/5/660.full#ref-list-1

This article cites 45 articles, 11 of which you can access for free at:

Citing articles

http://mcr.aacrjournals.org/content/12/5/660.full#related-urls

This article has been cited by 5 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/12/5/660To request permission to re-use all or part of this article, use this link


http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-13-0481http://mcr.aacrjournals.org/content/suppl/2014/02/27/1541-7786.MCR-13-0481.DC1http://mcr.aacrjournals.org/content/12/5/660.full#ref-list-1http://mcr.aacrjournals.org/content/12/5/660.full#related-urlshttp://mcr.aacrjournals.org/cgi/alertsmailto:[email protected]://mcr.aacrjournals.org/content/12/5/660http://mcr.aacrjournals.org/

TRAP1 Regulates Proliferation, Mitochondrial Function, and ......Cell Cycle and Senescence TRAP1...

Documents

Transcript of TRAP1 Regulates Proliferation, Mitochondrial Function, and ......Cell Cycle and Senescence TRAP1...