Published OnlineFirst February 24, 2014; DOI: 10.1158/1541-7786.MCR-13-0481
Molecular
Cancer
Research
Cell Cycle and Senescence
TRAP1 Regulates Proliferation, Mitochondrial Function, and
Has 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
Abstract
The 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 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 patients with NSCLC. These results
demonstrate that TRAP1 knockdown reduces cell growth and clonogenic cell survival. Moreover, TRAP1
downregulation 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 ¼ 0.008; multivariate analysis, HR: 2.554; 95% confidence interval, 1.085–6.012; P ¼ 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. Mol Cancer Res; 12(5); 1–10. 2014 AACR.
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
histologic subtypes: adenocarcinoma and squamous cell
carcinoma (SCC), accounting for 50% and 30% of all
Authors' Affiliations: 1Oncology Division, Center for Applied Medical
Research (CIMA), University of Navarra, Pamplona; 2Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo
Superior de Investigaciones Científicas, Madrid, Spain; 3Nuffield Department of Clinical Laboratory Sciences; 4Weatherall Institute of Molecular
Medicine, University of Oxford, John Radcliffe Hospital; 5Department of
Medical Oncology, University of Oxford, The Churchill Hospital, Oxford,
United Kingdom; 6Department of Rheumatology and Immunology, Shandong Provincial Hospital, Shandong University, Jinan, China; and 7Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale
Tumori, Milano, Italy
Note: Supplementary data for this article are available at Molecular Cancer
Research Online (http://mcr.aacrjournals.org/).
Corresponding Authors: Jackeline Agorreta, Oncology Division, Center
for 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]; and Francesco Pezzella, Nuffield Department of
Clinical Laboratory Sciences, University of Oxford, John Radcliffe 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.
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 tumor development and prognosis, is needed.
TNF receptor-associated protein 1 (TRAP1) is a mitochondrial protein that belongs to the 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 tumor 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 (10), the ATPase TBP7, a
component of the 19S proteasome regulatory subunit (11),
the Ca2þ-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 chemoresistance by blocking drug-induced apoptosis in a variety of tumors such as prostate cancer (18), osteosarcoma
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(15), and colorectal cancer (19). In addition, TRAP1 has
been reported to be upregulated in some tumors (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, retinoblastoma fails to inhibit proliferation (24).
However, the biologic role of this mitochondrial heat shock
protein (HSP) 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 TRAP1 in patients with NSCLC. 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 patients with NSCLC.
Materials and Methods
Cells
Human NSCLC cell lines NCI-A549 and NCI-H1299
were obtained from Clare Hall Laboratories and grown in
Dulbecco's Modified Eagle Medium supplemented with
10% FBS and penicillin-streptomycin at 100 U/mL. Cell
cultures were incubated at 37 C 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 (Navarra, Spain) from 2000 through 2008 were
included in this study. Clinicopathologic features of the
patients are listed in Table 1. Tumor specimens were
classified according to the 2004 World Health Organization criteria (25). The inclusion criteria were NSCLC
histology, no neoadjuvant chemo- or radiotherapy, and
absence of cancer within the 5 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 before
participation. 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
patients with NSCLC
Formalin-fixed paraffin-embedded tissue sections were
evaluated. Endogenous peroxidase activity was quenched
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Mol Cancer Res; 12(5) May 2014
Table 1. Clinicopathological characteristics of
the patients
N ¼ 71
Age, y (median–interquartile range)
<70
70
Sex, n (%)
Male
Female
Stage, n (%)
Stage I
Stage II
Stage III
Stage IV
Histology, n (%)
Adenocarcinoma
SCC
Other
Smoking status, n (%)
Current smoker
Former smoker
Nonsmoker
63 (54–70)
54 (76%)
17 (24%)
60 (85%)
11 (15%)
44 (62%)
15 (21%)
10 (14%)
2 (3%)
26 (37%)
39 (55%)
6 (8%)
39 (55%)
26 (37%)
6 (8%)
and antigen retrieval was carried out by pressure cooking
in 10 mmol/L citrate buffer, pH 6. Nonspecific binding
was blocked using 5% normal goat serum in Tris-buffered
saline for 30 minutes. Sections were incubated with antiTRAP1 antibody (1:400; Labvision) overnight at 4 C.
Sections were then incubated with Envision polymer
(Dako) for 30 minutes at room temperature. Peroxidase
activity was developed using diaminobenzidine and counterstained with hematoxylin before mounting in DPX
medium (BDH Chemical). The specificity of TRAP1
antibody was demonstrated using a variety of controls,
including Western blot analysis, inhibition with TRAP1siRNA sequences, isotype control, and omission of the
primary antibody.
Immunostaining evaluation
Two independent, blinded observers (F. Pezzella and
J. Agorreta) 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 percentage of positive cells
(0%–100%) and the intensity of staining (1þ, mild; 2þ,
moderate; and 3þ, intense labeling) were scored. Disagreements were resolved by common reevaluation.
Immunoblotting
Protein and total RNA were extracted using Paris kit
(Ambion-Life Technologies Ltd) according to the manufacturer's instructions. Thirty micrograms of total protein
from each lysate were boiled at 95 C for 5 minutes, separated
by SDS/PAGE under reduced conditions (5% 2-
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TRAP1 Regulates NSCLC Proliferation and Mitochondrial Function
mercaptoethanol), and transferred onto a nitrocellulose
membrane. The membranes were subsequently blocked in
5% defatted milk-PBS for 1 hour and incubated overnight at
4 C with a primary antibody anti TRAP1 (1:1000, Labvision) or anti b-actin (1:10000, Sigma). Blots were then
incubated with a horseradish peroxidase-linked secondary
antibody (1:5000; Amersham Pharmacia Biotech) and
developed by chemoluminiscence with Lumilight Plus Kit
(Roche diagnostics). Apoptosis detection by Western blotting was performed as described before (27).
RNA interference
For inhibition of TRAP1 expression, cells were seeded
(1 106 cells per well) in 10 cm dishes in antibiotic-free
medium. At 24 hours, cells were transfected with 40 nmol/L
of siRNA by using Oligofectamine (Invitrogen-Life Technologies Ltd) as previously described (21). Two siRNA
sequences against TRAP1 were designed and synthesized by
Eurogentec (TRAP1-siRNA1: 50 -AUGUUUGGAAGUGGAACCC-30 and 50 -ACCAUCUGAAAGCCACUGG-30 ;
TRAP1-siRNA2: 50 -TGCTGTTTGGAAGTGGAACCCTGCACGTTTTGGCCACTGACTGACGTGCAGGGCCACTTCCAAA-30 and 50 -CCTGTTTGGAAGTGGCCCTGCACGTCAGTCAGTGGCCAAAACGTGCAGGGTTCCACTTCCAAAC-30 ). A scrambled (scr) siRNA (50 AUGUUUGGAAGUGGAACCC-30 and 50 -UAGGGUGUACCCGUAAUAG-30 ) was used as the negative control.
RT-PCR
Retrotranscription was performed using RetroScript Kit
(Ambion). TRAP1 and b-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). 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
2DDCt method (28) using b-actin as endogenous gene and
scramble (scr) expression as calibrator.
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 mm sections
by pressure cooking in 10 mmol/L 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 minutes 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% dimethyl sulfoxide in FBS), fixed in 70% ethanol on ice, treated with 1
mg/mL RNase, stained with 10 mg/mL propidium iodide,
and examined with a FACSCalibur instrument fitted with a
Cell Quest software package (BD Biosciences). About
50,000 cells per sample were analyzed. Percentages of cells
in the Sub-G1, 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 mL of Annexin V (BD Biosciences) and 2 mL
of 10 mg/mL of propidium iodide (Sigma). Apoptosis was
induced by staurosporine treatment (1 mg/mL for 4 h).
Samples were analyzed on a FACSCalibur instrument and
quadrant analysis was performed with FlowJo 9.3 software
(Tree Star). At least three independent experiments per
condition were performed.
Clonogenic assay
Twenty four hours after siRNA transfection, cells were
harvested, seeded in triplicate (300 cells per well) in 6-well
plates, 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.
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. To analyze the mitochondrial membrane potential, TMRM (tetramethylrhodamine methylester; Invitrogen) staining was used because 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) was also
used 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) and fluorescence intensity was measured with ImageJ software (NIH,
Bethesda, MD).
Proliferation index determination
siRNA-treated cells were seeded in 10 cm dishes and
grown for 1, 3, or 5 days. Subsequently, cells were
Electron microscopy
Cells were fixed in 4% glutaraldehyde in 0.1 mol/L
phosphate buffer and processed for routine electron
Growth curves
Cells 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 Z2
particle count and size analyzer (Beckman Coulter). Automatically cell count was carried out with a Cell IQ microscope (Chipman Technologies).
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microscopy as previously described (32). Mitochondrial
mass was measured with ImageJ software.
Statistical analysis
Statistical analysis was performed using SPSS 15.0. Data
obtained from cell count, colony formation, MIB1 staining, cell cycle, and mitochondrial function experiments
were analyzed by the Student 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 analyze differences in
RFS (the median was selected as the cutoff value). Multivariate analysis was carried out using the Cox proportional hazards model. Only variables of P < 0.1 from the
univariate analysis were entered in the Cox regression
analysis. The proportional hazards assumption was
examined by testing interactions between the covariates
of the final model and time. P < 0.05 was considered
statistically significant.
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 real-time PCR (RT-PCR; Fig. 1A and
B). 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 (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 expression
by two independent TRAP1-siRNA sequences was demonstrated by RT-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, mean SD from at least three
independent experiments.
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TRAP1 Regulates NSCLC Proliferation and Mitochondrial Function
monitored by time-lapse video microscopy for 5 days at a
35- minute interval, confirming the reduction in cell
number after TRAP1 knockdown (Fig. 1D and Supplementary Movies S1 and S2). The impairment of cell survival
was further confirmed by clonogenic assay in H1299 and
A549 cell lines (Fig. 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 ki-67 protein (MIB1 antigen).
We found that from day 3, there was a significant reduction
of MIB1-positive cells when TRAP1 was inhibited (Fig. 2A).
Cell-cycle analysis by flow cytometry showed a significant
reduction in the percentage of cells in G2–M phase after
TRAP1 knockdown (Fig. 2B and C), confirming the
results from the immunohistochemical analysis of ki-67
expression.
TRAP1 downregulation 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 (Fig. 2D, top). Those effects were less evident in
H1299 cell line (Supplementary Fig. S1). The induction
of apoptosis in A549 cells was confirmed by the increase
of activated (cleaved) caspase-3, caspase-9, and PARP
(Fig. 2E, left). 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 (Fig. 2D, bottom
and Fig. 2E, right). The cell line H1299 did not show
clear evidence of apoptosis as silencing of TRAP produced
a 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 the
percentage of ki-67–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 staining was
performed on A549 cells transfected with scr, TRAP1-siRNA1, and TRAP1-siRNA2 sequences (top) or transfected cells treated with 1 mg/mL
þ
staurosporine (bottom) and analyzed by flow cytometry. Percentages of intact cells (Annexin V PI ), early apoptotic cells (Annexin V PI ) and late
þ
þ
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, mean SD from at least
three independent experiments.
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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 with 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, mean SD from three independent experiments.
cleaved caspase-9 while there was no evidence of caspase-3
activation (Supplementary Fig. S1).
No morphologic evidence of accumulation of apoptotic
bodies could be seen in the cell culture movies (Supplementary Movies).
TRAP1 downregulation impairs mitochondrial function
We hypothesized that the effects of TRAP1 inhibition
could be caused by mitochondrial dysfunction because
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 ¼ 0.002; Fig. 3A). Next, we examined
the effect of TRAP1 expression on mitochondrial membrane
potential as measured by TMRM uptake. A significant
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reduction on membrane potential was shown in TRAP1siRNA1–treated cells as compared with scr-siRNA (P <
0.001; Fig. 3B and C). However, no differences were found
in the mitochondrial mass measured by MitoTracker staining (P ¼ 0.809; Fig. 3D) or in ROS production measured by
MitoSOX staining (P ¼ 0.078; Fig. 3E). Electron microscopy also did not demonstrate changes in mitochondrial
morphology or mitochondrial mass (Fig. 3F).
High TRAP1 expression is associated with worse
prognosis in patients with NSCLC
TRAP1 expression was analyzed by immunohistochemistry in a series of 71 patients with NSCLC. The main clinical
and pathologic characteristics of these patients are summarized in Table 1. Moderate TRAP1 staining was found in
normal bronchial mucosa adjacent to the tumor (Fig. 4A),
although no immunoreactivity was found in alveoli (Fig.
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TRAP1 Regulates NSCLC Proliferation and Mitochondrial Function
B
C
D
E
% Recurrence-free survival
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
SCC of the lung (D). E, Kaplan–
Meier RFS curves for TRAP1
expression and log-rank test.
Shorter RFS time was found in
tumors bearing high TRAP1
expression. Scale bar, 50 mm.
A
100
Low expression
80
60
40
20
High expression
P = 0.008
0
0
20
40
60
80
100
120
Follow-up time (months)
4B). In tumor samples, TRAP1 staining was observed
predominantly in the cytoplasm of all tumors analyzed (Fig.
4C). In some cases, TRAP1 staining was both in the nucleus
and in the cytoplasm (Fig. 4D).
We next sought to evaluate the prognostic role of TRAP1
expression in our cohort of NSCLC. Nuclear expression of
TRAP1 did not correlate with prognosis (log-rank test; P ¼
0.486). However, when cytoplasmic TRAP1 expression was
analyzed, those patients with high TRAP1 expression (using
the median as the cutoff value) showed shorter RFS than
patients with low levels of TRAP1 (P ¼ 0.008; Fig. 4E).
Multivariate analysis using Cox regression model was performed to determine the independent prognostic factors.
Relevant clinicopathological variables such as age, gender,
stage, histology, and smoking history, as well as cytoplasmic
TRAP1 expression, were analyzed in the Cox univariate
model, and only those variables with a P value < 0.1 (stage
and TRAP1 expression) were included in the multivariate
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model. The Cox regression analysis revealed that high
cytoplasmic TRAP1 expression was an independent predictor of shorter RFS when patients were adjusted by stage [HR
¼ 2.554; confidence interval (CI), 1.085–6.012; Table 2].
Table 2. Multivariate Cox regression analysis of
RFS in patients with NSCLC
HR (95% CI)
Cytoplasmic TRAP1 expression
Low
—
High
2.554 (1.085–6.012)
Stage
I, II
—
III, IV
1.720 (0.725–4.077)
P
—
P ¼ 0.032
—
P ¼ 0.218
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These results demonstrate that TRAP1 expression correlates
with poor outcome in patients with NSCLC.
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 patients with NSCLC.
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 signaling (3, 4), it was then found
that TRAP1 also acts as a chaperon to retinoblastoma,
maintaining retinoblastoma protein in its active conformation (10). The importance of the role of TRAP1 as chaperon
has quickly outgrown its original role in retinoblastoma
as its pivotal role in cytoprotection has emerged. TRAP1
has been described to protect mitochondria from oxidative
stress and ROS (reviewed in refs. 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 tumor cells (38, 39). All these
functions are believed to be important for its role in protecting from apoptosis and inducing chemoresistance (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 antiapoptotic functions of
TRAP1 in NSCLC cell lines with clear 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 ROS (12, 14, 15, 18, 19). Having taken all these data into
account, we suggest that in many types of cells, loss of
TRAP1 has a mitochondrial priming effect which tips the
cells toward 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 pharmacologic stimulation, further
support the protective role of TRAP1 on apoptosis as its
downregulation can be the last step of the priming in some
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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
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 retinoblastoma 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 seems 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 retinoblastoma 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 and colleagues (40) although the potential suppressor effect in malignancies, 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, and
ovarian carcinoma (7, 19, 43, 45). Although 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 a matter of fact, a role as oncogene (40),
together with a role as tumor suppressor gene (44), has been
proposed. Our data obtained from lung primary tumors
showed both nuclear and cytoplasmic staining of TRAP1 in
tumor 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 tumor 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; ref. 46).
However, in a study on ovarian carcinoma, cytoplasmic
expression of TRAP1 was associated with better OS, whereas
Molecular Cancer Research
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Published OnlineFirst February 24, 2014; DOI: 10.1158/1541-7786.MCR-13-0481
TRAP1 Regulates NSCLC Proliferation and Mitochondrial Function
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 retinoblastoma
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
the shortest relapse-free survival in surgically resected
NSCLC. To our knowledge, this is the first study that
analyzes the role of TRAP1 in the prognosis of lung cancer.
Although the causes underlying diverse results in different
tumor types are still unknown, we may argue that TRAP1
plays organ-specific roles in each tumor 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 demonstrate a role in cell growth through the
regulation of the cell cycle. Furthermore, we have demonstrated that high TRAP1 expression is an adverse prognostic
factor for patients with NSCLC.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: J. Agorreta, J. Hu, A.L. Harris, K. Gatter, F. Pezzella
Development of methodology: J. Agorreta, J. Hu, H. Turley, F. Iborra, F. Pezzella
Acquisition of data (provided animals, acquired and managed patients, provided
facilities, etc.): J. Agorreta, J. Hu, D. Delia, D.J.P. Ferguson, F. Iborra, M.J. Pajares,
L.M. Montuenga, A.L. Harris, F. Pezzella
Analysis 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. Pezzella
Writing, 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. Pezzella
Administrative, technical, or material support (i.e., reporting or organizing data,
constructing databases): J. Agorreta, M. Larrayoz, K. Gatter, F. Pezzella
Study supervision: J. Agorreta, J. Hu, A.L. Harris, F. Pezzella
Acknowledgments
The authors thank G. Steers, Drs. R. Leek, K. Giaslakiotis, H. Mellor and
S. Wigfield for technical support.
Grant Support
This work was supported by Cancer Research UK grants and "UTE project
CIMA". J. Agorreta was funded by the Sara Borrell Program of ISCIII, Spanish
Government.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement
in 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.
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Molecular Cancer Research
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Published OnlineFirst February 24, 2014; DOI: 10.1158/1541-7786.MCR-13-0481
TRAP1 Regulates Proliferation, Mitochondrial Function, and
Has Prognostic Significance in NSCLC
Jackeline Agorreta, Jianting Hu, Dongxia Liu, et al.
Mol Cancer Res Published OnlineFirst February 24, 2014.
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