TRAP1 Regulates Proliferation, Mitochondrial Function and ......2014/02/22 · 2 Abstract The tumor...

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TRAP1 Regulates Proliferation, Mitochondrial Function and has Prognostic Significance in NSCLC

Jackeline Agorreta1,2, Jianting Hu2, Dongxia Liu2,3, Domenico Delia4, Helen Turley2, David J. P. Ferguson2, Francisco Iborra5,6, María J. Pajares1, Marta Larrayoz1, Isabel Zudaire1, Ruben Pio1, Luis M. Montuenga1, Adrian L. Harris5,7, Kevin Gatter2, Francesco Pezzella2

1Oncology Division, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain 2Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom 3Department of Rheumatology and Immunology, Shandong Provincial Hospital, Shandong University, Jinan, China 4Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Italy 5Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom 6Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain 7Department of Medical Oncology, University of Oxford, The Churchill Hospital, Oxford, United Kingdom

Running title: TRAP1 regulates NSCLC Proliferation and Mitochondrial Function

Keywords: TRAP1, non-small cell lung cancer, cell cycle, prognostic factor, mitochondrial function

Financial support: This work was supported by Cancer Research UK grants and “UTE project CIMA”.

JA was funded by the Sara Borrell Program of ISCIII, Spanish Government.

Corresponding authors: Jackeline Agorreta Oncology Division, Center for Applied Medical Research (CIMA), University of Navarra, Pio XII 55; 31008 Pamplona, Spain. Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Headley Way; OX3 9DU Oxford, United Kingdom. Work telephone: 34 948194700 Email address: [email protected]

Francesco Pezzella Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Headley Way; OX3 9DU Oxford, United Kingdom. Work telephone: 44 01865220497 Email address: [email protected]

Disclosure of potential conflicts of interest: No potential conflicts of interest are disclosed Manuscript word count: 3857 Number of tables: 2 Number of figures: 4

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

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 24, 2014; DOI: 10.1158/1541-7786.MCR-13-0481

http://mcr.aacrjournals.org/

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Abstract

The tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial heat shock

protein that has been related to drug resistance and protection from apoptosis in colorectal and

prostate cancer. Here the effect of TRAP1 ablation on cell proliferation, survival, apoptosis and

mitochondrial function was determined in non-small cell lung cancer (NSCLC). In addition, the

prognostic value of TRAP1 was evaluated in NSCLC patients. These results demonstrate that TRAP1

knockdown reduces cell growth and clonogenic cell survival. Moreover, TRAP1 down-regulation

impairs mitochondrial functions such as ATP production and mitochondrial membrane potential as

measured by TMRM (tetramethylrhodamine methylester) uptake, but it does not affect

mitochondrial density or mitochondrial morphology. The effect of TRAP1 silencing on apoptosis,

analyzed by flow cytometry and immunoblot 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 analysis which revealed that high TRAP1 expression was

associated with increased risk of disease recurrence (univariate analysis, P=0.008; multivariate

analysis, hazard ratio: 2.554; 95% CI: 1.085-6.012; P=0.03). In conclusion, these results demonstrate

that TRAP1 impacts the viability of NSCLC cells, and that its expression is prognostic in NSCLC.

Implications: TRAP1 controls NSCLC proliferation, apoptosis and mitochondrial function, and its

status has prognostic potential in NSCLC.

Abstract word count: 213




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Introduction

Lung cancer is the leading cause of cancer death worldwide (1). Non-small cell lung cancer (NSCLC),

the most common type of lung cancer, can be subdivided into two main histological subtypes:

adenocarcinoma (ADC) and squamous cell carcinoma (SCC), accounting for 50% and 30% of all

NSCLC cases, respectively (2). Despite the development of targeted therapies in lung cancer, there

has been little improvement in 5-year survival rates. In this context, improved knowledge of the

molecular biology of lung cancer, together with biomarkers that predict tumour development and

prognosis are needed.

Tumor necrosis factor (TNF) receptor associated protein 1 (TRAP1) is a mitochondrial protein that

belongs to the heat shock protein 90 (Hsp90) family, first identified as interacting with the

intracellular domain of the type I TNF receptor (3). Later sequence analysis revealed that TRAP1 was

identical to Hsp75 (4). TRAP1 is mainly localized in mitochondria of normal and tumour cells (4, 5)

acting as a substrate for the serine/threonine kinase PINK1 (6). Other localizations include the

cytosol, endoplasmic reticulum and nucleus (7-9). TRAP1 interacts with several proteins such as

retinoblastoma (RB) (10), the ATPase TBP7, a component of the 19S proteasome regulatory subunit

(11), the Ca2+-binding protein sorcin localized in the mitochondria (7, 12), the mitochondrial protein

cyclophilin D (5) and the tumor suppressors EXT1 and EXT2, proteins involved in hereditary multiple

exostoses (13). Moreover, TRAP1 has been reported to protect against apoptosis (5, 14, 15) and

oxidative stress (15-17). Interestingly, it has been proposed that TRAP1 may be involved in chemo-

resistance by blocking drug-induced apoptosis in a variety of tumours such as prostate cancer (18),

osteosarcoma (15) and colorectal cancer (19). In addition, TRAP1 has been reported to be up

regulated in some tumours (5, 18, 20) and downregulated in others (21). TRAP1 has been proposed

as a candidate biomarker in ovarian and prostate cancer (18, 22) and inhibition of TRAP1 is being

explored as a novel anticancer target (23). In NSCLC, we have previously demonstrated that TRAP1

positive cells have high levels of cell proliferation promoting genes (21), and that in the first hours

following hypoxia, in absence of TRAP1, RB fails to inhibits proliferation (24). However, the biological

role of this mitochondrial heat shock protein in NSCLC and its relation with mitochondrial 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 mitochondrial function in lung cancer cell lines and to evaluate the prognostic role of




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TRAP1 in NSCLC patients. Our results demonstrate that TRAP1 downregulation reduces cell

proliferation and survival, induces apoptosis and impairs mitochondrial functions such as ATP

production and mitochondrial membrane potential regulation. However, TRAP1 knockdown does

not affect mitochondrial density or mitochondrial morphology. In addition, overexpression of TRAP1

was associated with shorter recurrence-free survival (RFS) in NSCLC patients.

Material and Methods:

Cells

Human NSCLC cell lines NCI-A549 and NCI-H1299 were obtained from Clare Hall Laboratories

(London, UK) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10%

fetal bovine serum (FBS) and penicillin-streptomycin at 100 U/mL. Cell cultures were incubated at 37

ºC in a humidified 5% CO2 incubator.

Patient samples

A series of 71 patients with a diagnosis of NSCLC who underwent surgical resection at Clínica

Universidad de Navarra from 2000 through 2008 were included in this study. Clinicopathologic

features of the patients are listed in Table 1. Tumour specimens were classified according to the

2004 WHO criteria (25). The inclusion criteria were NSCLC histology, no neoadjuvant chemo- or

radio-therapy, and absence of cancer within the five years previous to lung cancer surgery. The study

protocol was approved by the institutional medical ethical committee. Written informed consent

was obtained from each patient prior to participation. Recurrence-free survival (RFS) was calculated

from the date of surgery to the date of detection of recurrence or the date of the last follow-up. The

median follow-up time was 42 months.

Immunohistochemistry in clinical specimens from NSCLC patients

Formalin-fixed paraffin-embedded tissue sections were evaluated. Endogenous peroxidase activity

was quenched and antigen retrieval was carried out by pressure cooking in 10 mM citrate buffer pH

6. Non-specific binding was blocked using 5% normal goat serum in Tris-buffered saline for 30 min.

Sections were incubated with anti-TRAP1 antibody (1:400; Labvision, Fremont, CA, USA) overnight at

4 ºC. Sections were then incubated with Envision polymer (Dako, Glostrup, Denmark) for 30 min at

room temperature. Peroxidase activity was developed using diaminobenzidine and counterstained

with hematoxylin before mounting in DPX medium (BDH Chemical, Poole, UK). The specificity of




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TRAP1 antibody was demonstrated using a variety of controls, including Western blot analysis,

inhibition with TRAP1-siRNA sequences, isotype control, and omission of the primary antibody.

Immunostaining evaluation

Two independent, blinded observers (FP and JA) evaluated the intensity and extensiveness of

staining in all of the study samples. The evaluation of cytoplasmic TRAP1 expression was performed

using the H-score system (26). Briefly, the percent of positive cells (0-100%) and the intensity of

staining (1+, mild; 2+, moderate and 3+, intense labeling) were scored. Disagreements were resolved

by common re-evaluation.

Immunoblotting

Protein and total RNA were extracted using Paris kit (Ambion-Life Technologies Ltd, Paisley, UK)

according to the manufacturer’s instructions. Thirty �g of total protein from each lysate were boiled

at 95 ºC for 5 min, separated by SDS/PAGE under reduced conditions (5% 2-mercaptoethanol) and

transferred onto a nitrocellulose membrane. The membranes were subsequently blocked in 5%

defatted milk-PBS for 1 h and incubated overnight at 4ºC with a primary antibody anti TRAP1

(1:1000, Labvision) or anti β-actin (1:10000, Sigma, Dorset, UK). Blots were then incubated with a

horseradish peroxidase-linked secondary antibody (1:5000; Amersham Pharmacia Biotech, Little

Chalfont, UK) and developed by chemoluminiscence with Lumilight plus kit (Roche diagnostics,

Burgess Hill, UK). Apoptosis detection by western blotting was performed as described before (27).

RNA interference

For inhibition of TRAP1 expression, cells were seeded (1 x 106 cells per well) in 10 cm dishes in

antibiotic-free medium. At 24 h, cells were transfected with 40 nM of siRNA by using Oligofectamine

(Invitrogen-Life Technologies Ltd, Paisley, UK) as previously described (21). Two siRNA sequences

against TRAP1 were designed and synthesized by Eurogentec (Eurogentec, Southampton, UK)

(TRAP1-siRNA1: 5’-AUGUUUGGAAGUGGAACCC-3’ and 5’-ACCAUCUGAAAGCCACUGG-3’; TRAP1-

siRNA2: 5’-TGCTGTTTGGAAGTGGAACCCTGCACGTTTTGGCCACTGACTGACGTGCAGGGCCACTTCCAAA-

3´ and 5´-CCTGTTTGGAAGTGGCCCTGCACGTCAGTCAGTGGCCAAAACGTGCAGGGTTCCACTTCCAAAC-

3´). A scrambled (scr) siRNA (5’-AUGUUUGGAAGUGGAACCC-3’ and 5’-UAGGGUGUACCCGUAAUAG-

3’) was used as the negative control.




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RT-PCR

Retrotranscription was performed using RetroScript kit (Ambion). TRAP1 and β-actin expression was

analyzed by PCR using TaqMan Gene Expression Assays (Applied Biosystems). The reaction was

performed on a PTC-200 thermal cycler with a Chromo 4 continuous fluorescence detector (Bio-Rad,

Hemel Hempstead, UK). The comparative cycle threshold (CT) method was used to analyze the data

by generating relative values of the amount of target cDNA, according to the 2-��CT method (28) using

�-actin as endogenous gene and scramble (scr) expression as calibrator.

Growth curves

Cells were seeded on six-well dishes at a density of 1 x 105 cells per well in triplicate and exposed to

normoxia for 1 to 7 days. Subsequently, cell number was assessed with a Coulter Z2 particle count

and size analyzer (Beckman Coulter, High Wycombe, UK). Automatically cell count was carried out

with a Cell IQ microscope (Chipman Technologies, Tampere, Finland).

Clonogenic assay

Twenty four hours after siRNA transfection, cells were harvested, seeded in triplicate (300 cells per

well) in six-well plates, and incubated at 37 ºC 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 determination

siRNA 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-buffered formalin (pH 7.0), suspended in agar

and embedded in paraffin. Antigen retrieval was carried out in 3 μm sections by pressure cooking in

10 mM citrate buffer pH 6, and immunohistochemical staining for the human Ki-67 protein was

performed using the anti-MIB1 antigen antibody (Dako) at 1:50 for 30 min at room temperature.

Sections were incubated with the Envision detection system (Dako) and developed with

diaminobenzidine. Immunohistochemical scoring was performed as previously described (29).

Cell cycle and apoptosis analysis

Cell cycle analyses were performed on trypsin-disaggregated cryopreserved cell suspensions

containing floating and attached cells. Following thawing, cells were centrifuged to remove the

cryopreservation solution (10% DMSO in FBS), fixed in 70% ethanol on ice, treated with 1 μg/ml




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RNase, stained with 10 μg/ml propidium iodide and examined with a FACSCalibur instrument fitted

with a Cell Quest software package (BD Biosciences, Sunnyvale, CA, USA). About 50,000 cells per

sample were analyzed. Percentages of cells in the SubG1, G1, S and G2/M phases were determined.

For apoptosis analysis fresh trypsin-disaggregated cell suspensions containing floating and attached

cells were used as previously described (30). Briefly, cells were washed and stained with 2 �l of

annexin V (BD Biosciences) and 2 �l of 10 �g/ml of propidium iodide (Sigma). Samples were analyzed

on a FACSCalibur instrument and quadrant analysis was performed with FlowJo 9.3 software (Tree

Star, Ashland, OR, USA). At least three independent experiments per condition were performed.

Mitochondrial function

The amount of ATP was measured in lysates of 105 cells using the ATP Bioluminescence Assay Kit

(Roche) in accordance with the manufacturer's instructions. This method uses the ATP dependency

of the light-emitting, luciferase-catalyzed oxidation of luciferin for the measurement of ATP

concentration. In order to analyze the mitochondrial membrane potential, TMRM

(tetramethylrhodamine methyl ester; Invitrogen) staining was used since it is a cell-permeant,

cationic, red-orange fluorescent dye that is readily sequestered by active mitochondria. MitoTracker

Green staining (Molecular Probes-Life Technologies Ltd, Paisley, UK) was also used in order to

measure mitochondrial mass regardless of mitochondrial membrane potential. Moreover, the

production of reactive oxygen species (ROS) was evaluated by the MitoSOX staining (Molecular

probes) as previously described (31). Fluorescence images were collected using a confocal

microscope (Zeiss LSM 510 META, Carl Zeiss, Cambridge, UK) and fluorescence intensity was

measured with ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).

Electron microscopy

Cells were fixed in 4% glutaraldehyde in 0.1 M phosphate buffer and processed for routine electron

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

Statistical analysis

Statistical analysis was performed using SPSS 15.0 (Chicago, IL, USA). Data obtained from cell count,

colony formation, MIB1 staining, cell cycle and mitochondrial function experiments were analyzed

by the Student´s t test or the Mann-Whitney U test for parametric and nonparametric variables,

respectively. For survival analysis, Kaplan-Meier survival curves and the log rank test were used to




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analyze differences in RFS (the median was selected as the cut-off value). Multivariate analysis was

carried out using the Cox proportional hazards model. Only variables of P

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Results

Expression of TRAP1 is necessary for cell growth

To examine the effect of TRAP1 inhibition on cell proliferation, we carried out downregulation

experiments in lung cancer cell lines. Knockdown was carried out in H1299 and A549 cells using two

different siRNAs and the efficacy of TRAP1 siRNA downregulation was verified by Western blotting

and RT-PCR (Figures 1A and 1B). TRAP1 downregulation resulted in a significant reduction in cell

growth in both H1299 and A549 cell lines as confirmed by TRAP1-siRNA1 and 2 sequences (Figure

1C). Cell growth of TRAP1-siRNA1 treated A549 cells was also monitored by time-lapse video

microscopy for 5 days at a 35 min interval, confirming the reduction in cell number after TRAP1

knockdown (Figure 1D; Supplementary Movies 1 and 2). The impairment of cell survival was further

confirmed by clonogenic assay in H1299 and A549 cell lines (Figure 1E). We next investigated the

effect of TRAP1 knockdown on cell proliferation by staining cell pellets of scr- and TRAP1-siRNA A549

treated cells at different time points for ki67 protein (MIB1 antigen). We found that from day 3 there

was a significant reduction of MIB1 positive cells when TRAP1 was inhibited (Figure 2A). Cell cycle

analysis by flow cytometry showed a significant reduction in the percentage of cells in G2/M phase

after TRAP1 knockdown (Figures 2B and 2C), confirming the results from the immunohistochemical

analysis of ki67 expression.

TRAP1 down regulation has a variable effect on apoptosis

Quantification of apoptotic cells by Annexin V/PI assay showed that TRAP1 downregulated A549

cells had increased apoptotic rates as compared with scr-siRNA treated (Figure 2D, top panel). Those

effects were less evident in H1299 cell line (Supplementary Figure 1). The induction of apoptosis in

A549 cells was confirmed by the increase of activated (cleaved) caspase 3, caspase 9 and PARP

(Figure 2E, left panel). It should be noted that when apoptosis was induced by treating cells with

staurosporine, there was a dramatic induction of apoptosis in TRAP1-siRNA treated cells but not in

scr-control cells (Figure 2D bottom panel and Figure 2E right panel). The cell line H1299 did not show

clear evidences of apoptosis as silencing of TRAP produced a mild increase of PARP but a similarly

mild decrease of cleaved caspase 9 while there were no evidences of caspase 3 activation

(Supplementary Figure 1).




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No morphological evidences of accumulation of apoptotic bodies could be seen in the cell culture

movies (Supplementary Movie).

TRAP1 downregulation impairs mitochondrial function

We hypothesized that the effects of TRAP1 inhibition could be caused by mitochondrial dysfunction,

since TRAP1 is known to be mainly expressed in the mitochondria. Therefore, we analyzed a variety

of mitochondrial functions after TRAP1 inhibition. First, we analyzed ATP production in scr- and

TRAP1-siRNA1 treated cells, showing that TRAP1 inhibition was associated with a 30% reduction of

ATP (P=0.002; Figure 3A). Next, we examined the effect of TRAP1 expression on mitochondrial

membrane potential as measured by TMRM uptake. A significant reduction on membrane potential

was shown in TRAP1-siRNA1 treated cells as compared to scr-siRNA (P

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that high cytoplasmic TRAP1 expression was an independent predictor of shorter RFS when patients

were adjusted by stage [HR=2.554; CI (1.085–6.012)] (Table 2). These results demonstrate that

TRAP1 expression correlates with poor outcome in NSCLC patients.

Discussion

In the present study, we have demonstrated that TRAP1 has important effects on mitochondrial

function and plays a key role in the regulation of proliferation, survival and apoptosis of NSCLC cells.

Moreover, we have shown that high cytoplasmic TRAP1 expression is associated with worse

prognosis in NSCLC patients.

During the last 10 years an increasing number of studies have demonstrated the versatility of TRAP1

protein and its involvement in a number of pathways. Originally cloned because of its interaction

with TNF receptor, and therefore likely to be involved in cell signalling (3, 4), it was then found that

TRAP1 also acts as a chaperon to Retinoblastoma (RB), maintaining RB protein in its active

conformation (10). The importance of the role of TRAP1 as chaperon has quickly outgrown its

original role in RB as its pivotal role in cytoprotection has emerged. TRAP1 has been described to

protect mitochondria from oxidative stress and reactive oxygen species (ROS) [reviewed at (33-35)].

In this sense, TRAP1 blocks ROS activity (15), ROS production (36) and regulates the mitochondrial

permeability transition pores (5, 37). It has been recently demonstrated that TRAP1 has an

important role controlling central metabolic networks in the mitochondria of tumour cells (38, 39).

All these functions are believed to be important for its role in protecting from apoptosis and

inducing chemo-resistance (11, 33). In the present study, we investigated the role of TRAP1 in NSCLC

cell lines growth by looking at its effects on cell proliferation, apoptosis and mitochondrial function.

Downregulation of TRAP1 expression in NSCLC cell lines produced a significant reduction of cell

proliferation and survival as assessed by cell count, clonogenic assays, ki67 expression and cell cycle

analysis. In agreement with these results, proliferation had been previously linked to TRAP1 by our

group (24) and others (40). However, there is no general agreement in literature as some authors

have failed to notice any effect on cell growth (38). Moreover, we have determined that TRAP1

knockdown is associated with a subtle and variable effect on induction of apoptosis. This induction

becomes more evident when apoptosis is induced pharmacologically with staurosporine. Our results

have demonstrated a variable role in anti-apoptotic functions of TRAP1 in NSCLC cell lines with clear




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signs of apoptosis detected in one of the cell lines studied. These results are still in agreement with

previous reports showing a protective effect of TRAP1 against apoptosis-inducing anticancer drugs

or reactive oxygen species (12, 14, 15, 18, 19). Having taken all these data into account, we suggests

that in many types of cells, loss of TRAP1 has a mitochondrial priming effect which tips the cells

towards apoptosis making them more sensitive to the effects of cytotoxic drugs (41, 42). The

observation that apoptosis is obvious in one of the two cell lines analyzed, independently from the

pharmacological stimulation, further support the protective role of TRAP1 on apoptosis as its

downregulation 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 and of the mitochondrial membrane potential. These findings are consistent with

previous results indicating that TRAP1 in cancer prevents mitochondrial damage (33) and after its

inhibition, the misfolding of Peptidylprolyl isomerases D results in loss of ATP production (5) and are

also consistent with its protective role from apoptosis. The role of TRAP1 in maintaining ATP levels

has also been reported in rat brain (43). In contrast, two recent papers (40, 44) report that loss of

TRAP1 results in an increase of ATP production following a switch from glycolysis to oxidative

phosphorylation. We cannot provide an explanation for this discrepancy as far as the role of TRAP1

in ATP production is concerned, but it is likely to be with the different types of final effects that

TRAP1 can have according to the type of cell being investigated.

We have previously demonstrated that after a short hypoxic shock, TRAP1 translocates to the

nucleus and is essential to maintain RB suppressor gene function: in its absence, the cells fail to slow

down proliferation (24). However this is a short term effect and, in agreement with our previous

results and as demonstrated here, in normoxia and in the longer term TRAP1 promotes the cell

cycle. Therefore it appears to have two opposite functions: in normoxia, TRAP1 promotes cell

proliferation and protects from apoptosis, however following a hypoxic shock it moves to the

nucleus where it is essential for RB to induce a rapid, short term slowing down of proliferation. If

hypoxia persists, TRAP1 cytoplasmic levels will decrease (unpublished results) alongside a diminution

of the proliferation rate. In this respect, it can be considered to have oncogenic properties as

suggested by Sciacovelli et al (40) although the potential suppressor effect in malignancies, due to its

chaperone role to RB (10), remains to be elucidated.




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On the other hand, TRAP1 expression has also been correlated with chemorresistance in breast,

colorectal and ovarian carcinoma (7, 19, 43, 45). While there is consensus that TRAP1 expression is a

predictive biomarker for drug resistance, because of the broad range of cellular functions influenced

by TRAP1 (33), it is perhaps not surprising that its role as immunohistochemical prognostic

biomarker is controversial. As matter of fact, a role as oncogene (40), together with a role as tumour

suppressor gene (44) have been proposed. Our data obtained from lung primary tumours showed

both nuclear and cytoplasmic staining of TRAP1 in tumour cells, as it was previously reported in

NSCLC, breast carcinoma, ovarian cancer and other malignancies (18, 24, 45). Only a few studies

have been performed looking at TRAP1 expression on tumour samples and correlations with

prognosis (24, 45, 46), and the results are controversial. In colorectal carcinoma, high TRAP1

expression has been correlated with shorter RFS and overall survival (OS) (46). However, in a study

on ovarian carcinoma, cytoplasmic expression of TRAP1 was associated with better OS while no

association was found with RFS (45). Finally, in a series of breast carcinoma we found no correlation

between cytoplasmic TRAP1 and OS or RFS, although nuclear TRAP1 expression was instead

associated with both RB positivity and longer RFS but no association was found with OS (24). In the

present study we demonstrated that cytoplasmic expression of TRAP1 is an independent predictor

of shortest relapse-free survival in surgically resected NSCLC. To our knowledge, this is the first study

that analyses the role of TRAP1 in the prognosis of lung cancer. Although the causes underlying

diverse results in different tumour types are still unknown, we may argue that TRAP1 plays organ-

specific roles in each tumour type. Indeed, it should be noted that the association between

cytoplasmic staining and worse prognosis demonstrated in NSCLC in the present study and in

colorectal carcinoma (46) is fully consistent with the suggested role of TRAP1 in cell proliferation and

protection to apoptosis. However, more extensive studies exploring the role of TRAP1 as a potential

target or predictor 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

explanation for the discrepant results observed in literature; however our data further support the

role played by TRAP1 in mitochondrial function and regulation of apoptosis but also demonstrates a

role in cell growth through the regulation of cell cycle. Furthermore, we have demonstrated that

high TRAP1 expression is an adverse prognostic factor for NSCLC patients.




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List of abbreviations: adenocarcinoma (ADC); fetal bovine serum (FBS); heat shock protein (Hsp);

non-small cell lung cancer (NSCLC); reactive oxygen species (ROS); recurrence-free survival (RFS);

retinoblastoma (RB); squamous cell carcinoma (SCC); tetramethylrhodamine methyl ester (TMRM);

tumor necrosis factor receptor-associated protein 1 (TRAP1).

Authors' contributions: FP, AH and KG planned and supervised the study. JA, JH, DL, HT and AI

performed experimental work. DD and DF provided technical expertise in apoptosis detection and

electron microscopy techniques, respectively. FI performed the live-imaging experiments and

measurement of fluorescence intensities. MJP, ML and IZ helped with the evaluation of TRAP1

expression on tumour growth. RP and LMM participated in the collection of human samples and the

evaluation of the prognostic role of TRAP1. JA and FP did the immunohistochemical evaluation of

TRAP1 expression and wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements: Authors thank Mr. G. Steers, Dr. R. Leek, Dr. K. Giaslakiotis, Dr. H. Mellor and

Dr. S. Wigfield for technical support.




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TABLES:

Table 1: Clinicopathological characteristics of the patients

N= 71 Age-years (median-interquartile range) 63 (54-70)

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Table legends:

Table 1: Clinicopathological characteristics of the patients

Table 2: Multivariate Cox regression analysis of RFS in patients with NSCLC

Figure legends:

Figure 1: TRAP1 knockdown inhibits cell proliferation and survival on the H1299 and A549 cell lines. Successful knockdown of TRAP1 expression by two independent TRAP1-siRNA sequences was demonstrated by real-time PCR (A) and Western blot analysis (B) in both H1299 and A549 cell lines at day 4. To determine the effect of TRAP1 siRNA knockdown on tumor cell proliferation, cells were transfected with control- (scr) or TRAP1-siRNAs and cell number was determined by a Coulter Z2 particle count and size analyzer (C) or automatically determined by a Cell IQ microscope (D). (E) TRAP1 downregulation significantly reduced colony formation in the A549 and H1299 cell lines. Data are presented as mean ± standard deviation from at least three independent experiments. Figure 2: Downregulation of TRAP1 arrests cell proliferation and induces apoptosis in the A549 lung cancer cell line. (A) Proliferative fraction given by the percentage of ki67 positive cells was significantly reduced in TRAP1-siRNA1 treated cells. (B) Cell cycle distribution of A549 cells at different days after TRAP1-siRNA1 transfection. (C) Differences in the percentage of cells in S and G2/M phases at day 3. (D) Annexin V/propidium iodide (PI) staining was performed on A549 cells transfected with scr, TRAP1-siRNA1 and TRAP1-siRNA2 sequences (top panel) or transfected cells treated with 1 μg/ml staurosporine (bottom panel) and analyzed by flow cytometry. Percentages of intact cells (Annexin V� PI�), early apoptotic cells (Annexin V+ PI�) and late apoptotic or necrotic cells (Annexin V+ PI+) 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 are presented as mean ± standard deviation from at least three independent experiments. Figure 3: TRAP1 downregulation impairs mitochondrial function in A549 cells. (A) Measurement of ATP levels by a bioluminescence assay demonstrates that TRAP1 inhibition reduces ATP levels. (B) Representative images of TMRM uptake in scr- and TRAP1-siRNA1 treated cells. (C) Quantification of TMRM uptake by image analysis shows a reduction in mitochondrial membrane potential in TRAP1-siRNA1 treated A549 cells as compared to control cells. (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 no differences in the mitochondrial morphology were observed between scr- and TRAP1-siRNA1 treated cells. Moreover, no significant differences in mitochondrial mass were found. Scale bar, 500 nm. Data are presented as mean ± standard deviation from three independent experiments.




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Figure 4: High cytoplasmic TRAP1 staining is associated with adverse prognosis in NSCLC. Representative TRAP1 immunostaining in bronchial epithelium (A), lung parenchyma (B), lung adenocarcinoma (C), and squamous cell carcinoma of the lung (D). (E) Kaplan Meier recurrence-free survival curves for TRAP1 expression and log-rank test. Shorter recurrence-free survival time was found in tumours bearing high TRAP1 expression. Scale bar, 50 μm.




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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

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