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.
Keywords: TRAP1, non-small cell lung cancer, cell cycle, prognostic factor, mitochondrial function
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 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.
Table 1. Clinicopathological characteristics of the patients.
N= 71 | |
---|---|
Age-years (median-interquartile range) | 63 (54-70) |
<70 | 54 (76%) |
≥70 | 17 (24%) |
Sex-n (%) | |
Male | 60 (85%) |
Female | 11 (15%) |
Stage-n (%) | |
Stage I | 44 (62%) |
Stage II | 15 (21%) |
Stage III | 10 (14%) |
Stage IV | 2 (3%) |
Histology-n (%) | |
Adenocarcinoma | 26 (37%) |
Squamous cell carcinoma | 39 (55%) |
Other | 6 (8%) |
Smoking status-n (%) | |
Current smoker | 39 (55%) |
Former smoker | 26 (37%) |
Non-smoker | 6 (8%) |
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 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 μg 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 × 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.
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-ΔΔCT method (28) using β-actin as endogenous gene and scramble (scr) expression as calibrator.
Growth curves
Cells were seeded on six-well dishes at a density of 1 × 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 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 μl of annexin V (BD Biosciences) and 2 μl of 10 μg/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 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<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. A P value less than 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 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.
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.
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).
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<0.001; Figures 3B and 3C). However, no differences were found in the mitochondrial mass measured by MitoTracker staining (P=0.809, Figure 3D) or in ROS production measured by MitoSOX staining (P=0.078; Figure 3E). Electron microscopy also did not demonstrate changes in mitochondrial morphology or mitochondrial mass (Figure 3F).
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.
High TRAP1 expression is associated with worse prognosis in NSCLC patients
TRAP1 expression was analysed by immunohistochemistry in a series of 71 NSCLC patients. The main clinical and pathological characteristics of these patients are summarized in Table 1. Moderate TRAP1 staining was found in normal bronchial mucosa adjacent to the tumour (Figure 4A), although no immunoreactivity was found in alveoli (Figure 4B). In tumour samples, TRAP1 staining was observed predominantly in the cytoplasm of all tumours analyzed (Figure 4C). In some cases, TRAP1 staining was both in the nucleus as well as in the cytoplasm (Figure 4D).
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.
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=0.486). However, when cytoplasmic TRAP1 expression was analyzed, those patients with high TRAP1 expression (using the median as the cut off value) showed shorter recurrence-free survival (RFS) than patients with low levels of TRAP1 (P=0.008; Figure 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 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=2.554; CI (1.085–6.012)] (Table 2). These results demonstrate that TRAP1 expression correlates with poor outcome in NSCLC patients.
Table 2. Multivariate Cox regression analysis of RFS in patients with NSCLC.
Hazard ratio (95% confidence interval) | P value | |
---|---|---|
Cytoplasmic TRAP1 expression | ||
Low | ||
High | 2.554 (1.085-6.012) | P=0.032 |
Stage | ||
I, II | ||
III, IV | 1.720 (0.725-4.077) | P=0.218 |
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 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.
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.
Supplementary Material
Acknowledgements
Authors thank Mr. G. Steers, Dr. R. Leek, Dr. K. Giaslakiotis, Dr. H. Mellor and Dr. S. Wigfield for technical support.
List of abbreviations
- (ADC)
adenocarcinoma
- (FBS)
fetal bovine serum
- (Hsp)
heat shock protein
- (NSCLC)
non-small cell lung cancer
- (ROS)
reactive oxygen species
- (RFS)
recurrence-free survival
- (RB)
retinoblastoma
- (SCC)
squamous cell carcinoma
- (TMRM)
tetramethylrhodamine methyl ester
- (TRAP1)
tumor necrosis factor receptor-associated protein 1
Footnotes
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.
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.
Disclosure of potential conflicts of interest: No potential conflicts of interest are disclosed
References
- 1.Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63:11–30. doi: 10.3322/caac.21166. [DOI] [PubMed] [Google Scholar]
- 2.Perez-Moreno P, Brambilla E, Thomas R, Soria JC. Squamous cell carcinoma of the lung: molecular subtypes and therapeutic opportunities. Clin Cancer Res. 2012;18:2443–51. doi: 10.1158/1078-0432.CCR-11-2370. [DOI] [PubMed] [Google Scholar]
- 3.Song HY, Dunbar JD, Zhang YX, Guo D, Donner DB. Identification of a protein with homology to hsp90 that binds the type 1 tumor necrosis factor receptor. J Biol Chem. 1995;270:3574–81. [PubMed] [Google Scholar]
- 4.Felts SJ, Owen BA, Nguyen P, Trepel J, Donner DB, Toft DO. The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties. J Biol Chem. 2000;275:3305–12. doi: 10.1074/jbc.275.5.3305. [DOI] [PubMed] [Google Scholar]
- 5.Kang BH, Plescia J, Dohi T, Rosa J, Doxsey SJ, Altieri DC. Regulation of tumor cell mitochondrial homeostasis by an organelle-specific Hsp90 chaperone network. Cell. 2007;131:257–70. doi: 10.1016/j.cell.2007.08.028. [DOI] [PubMed] [Google Scholar]
- 6.Pridgeon JW, Olzmann JA, Chin LS, Li L. PINK1 protects against oxidative stress by phosphorylating mitochondrial chaperone TRAP1. PLoS Biol. 2007;5:e172. doi: 10.1371/journal.pbio.0050172. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Maddalena F, Sisinni L, Lettini G, Condelli V, Matassa DS, Piscazzi A, et al. Resistance to paclitxel in breast carcinoma cells requires a quality control of mitochondrial antiapoptotic proteins by TRAP1. Mol Oncol. 2013 doi: 10.1016/j.molonc.2013.04.009. S1574-7891(13)00079-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Cechetto JD, Gupta RS. Immunoelectron microscopy provides evidence that tumor necrosis factor receptor-associated protein 1 (TRAP-1) is a mitochondrial protein which also localizes at specific extramitochondrial sites. Exp Cell Res. 2000;260:30–9. doi: 10.1006/excr.2000.4983. [DOI] [PubMed] [Google Scholar]
- 9.Takemoto K, Miyata S, Takamura H, Katayama T, Tohyama M. Mitochondrial TRAP1 regulates the unfolded protein response in the endoplasmic reticulum. Neurochem Int. 2011;58:880–7. doi: 10.1016/j.neuint.2011.02.015. [DOI] [PubMed] [Google Scholar]
- 10.Chen CF, Chen Y, Dai K, Chen PL, Riley DJ, Lee WH. A new member of the hsp90 family of molecular chaperones interacts with the retinoblastoma protein during mitosis and after heat shock. Mol Cell Biol. 1996;16:4691–9. doi: 10.1128/mcb.16.9.4691. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Amoroso MR, Matassa DS, Laudiero G, Egorova AV, Polishchuk RS, Maddalena F, et al. TRAP1 and the proteasome regulatory particle TBP7/Rpt3 interact in the endoplasmic reticulum and control cellular ubiquitination of specific mitochondrial proteins. Cell Death Differ. 2012;19:592–604. doi: 10.1038/cdd.2011.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Landriscina M, Laudiero G, Maddalena F, Amoroso MR, Piscazzi A, Cozzolino F, et al. Mitochondrial chaperone Trap1 and the calcium binding protein Sorcin interact and protect cells against apoptosis induced by antiblastic agents. Cancer Res. 2010;70:6577–86. doi: 10.1158/0008-5472.CAN-10-1256. [DOI] [PubMed] [Google Scholar]
- 13.Simmons AD, Musy MM, Lopes CS, Hwang LY, Yang YP, Lovett M. A direct interaction between EXT proteins and glycosyltransferases is defective in hereditary multiple exostoses. Hum Mol Genet. 1999;8:2155–64. doi: 10.1093/hmg/8.12.2155. [DOI] [PubMed] [Google Scholar]
- 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. doi: 10.1074/jbc.M404256200. [DOI] [PubMed] [Google Scholar]
- 15.Montesano Gesualdi N, Chirico G, Pirozzi G, Costantino E, Landriscina M, Esposito F. Tumor necrosis factor-associated protein 1 (TRAP-1) protects cells from oxidative stress and apoptosis. Stress. 2007;10:342–50. doi: 10.1080/10253890701314863. [DOI] [PubMed] [Google Scholar]
- 16.Im CN, Lee JS, Zheng Y, Seo JS. Iron chelation study in a normal human hepatocyte cell line suggests that tumor necrosis factor receptor-associated protein 1 (TRAP1) regulates production of reactive oxygen species. J Cell Biochem. 2007;100:474–86. doi: 10.1002/jcb.21064. [DOI] [PubMed] [Google Scholar]
- 17.Voloboueva LA, Duan M, Ouyang Y, Emery JF, Stoy C, Giffard RG. Overexpression of mitochondrial Hsp70/Hsp75 protects astrocytes against ischemic injury in vitro. J Cereb Blood Flow Metab. 2008;28:1009–16. doi: 10.1038/sj.jcbfm.9600600. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Leav I, Plescia J, Goel HL, Li J, Jiang Z, Cohen RJ, et al. Cytoprotective mitochondrial chaperone TRAP-1 as a novel molecular target in localized and metastatic prostate cancer. Am J Pathol. 2010;176:393–401. doi: 10.2353/ajpath.2010.090521. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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 colorectal carcinoma cells. Cancer Lett. 2009;279:39–46. doi: 10.1016/j.canlet.2009.01.018. [DOI] [PubMed] [Google Scholar]
- 20.Fang W, Li X, Jiang Q, Liu Z, Yang H, Wang S, et al. Transcriptional patterns, biomarkers and pathways characterizing nasopharyngeal carcinoma of Southern China. J Transl Med. 2008;6:32. doi: 10.1186/1479-5876-6-32. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Liu D, Hu J, Agorreta J, Cesario A, Zhang Y, Harris AL, et al. Tumor necrosis factor receptor-associated protein 1(TRAP1) regulates genes involved in cell cycle and metastases. Cancer Lett . 2010;296:194–205. doi: 10.1016/j.canlet.2010.04.017. [DOI] [PubMed] [Google Scholar]
- 22.Landriscina M, Amoroso MR, Piscazzi A, Esposito F. Heat shock proteins, cell survival and drug resistance: the mitochondrial chaperone TRAP1, a potential novel target for ovarian cancer therapy. Gynecol Oncol . 2010;117:177–82. doi: 10.1016/j.ygyno.2009.10.078. [DOI] [PubMed] [Google Scholar]
- 23.Siegelin MD. Inhibition of the mitochondrial Hsp90 chaperone network: a novel, efficient treatment strategy for cancer? Cancer Lett. 2013;333:133–46. doi: 10.1016/j.canlet.2013.01.045. [DOI] [PubMed] [Google Scholar]
- 24.Hu J, Tan EY, Campo L, Leek R, Seman Z, Turley H, et al. TRAP1 is involved in cell cycle regulated by retinoblastoma susceptibility gene (RB1) in early hypoxia and has variable expression patterns in human tumors. Journal of Cancer Research Updates. 2013 in press. [Google Scholar]
- 25.Travis WD, Brambilla E, Müller-Hermelink HK, Harris CC. Tumours of the Lung, Pleura, Thymus and Heart. Lyon, France: IARC Press; 2004. [Google Scholar]
- 26.Pajares MJ, Agorreta J, Larrayoz M, Vesin A, Ezponda T, Zudaire I, et al. Expression of tumor-derived vascular endothelial growth factor and its receptors is associated with outcome in early squamous cell carcinoma of the lung. J Clin Oncol. 2012;30:1129–36. doi: 10.1200/JCO.2011.37.4231. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Cossu F, Milani M, Vachette P, Malvezzi F, Grassi S, Lecis D, et al. Structural insight into inhibitor of apoptosis proteins recognition by a potent divalent smac-mimetic. PLoS One. 2012;7:e49527. doi: 10.1371/journal.pone.0049527. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–8. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
- 29.Sington J, Giatromanolaki A, Campo L, Turley H, Pezzella F, Gatter KC. BNIP3 expression in follicular lymphoma. Histopathology. 2007;50:555–60. doi: 10.1111/j.1365-2559.2007.02657.x. [DOI] [PubMed] [Google Scholar]
- 30.de Miguel FJ, Sharma RD, Pajares MJ, Montuenga LM, Rubio A, Pio R. Identification of alternative splicing events regulated by the oncogenic factor SRSF1 in lung cancer. Cancer Res. 2013 doi: 10.1158/0008-5472.CAN-13-1481. [DOI] [PubMed] [Google Scholar]
- 31.das Neves RP, Jones NS, Andreu L, Gupta R, Enver T, Iborra FJ. Connecting variability in global transcription rate to mitochondrial variability. PLoS Biol. 2010;8:e1000560. doi: 10.1371/journal.pbio.1000560. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Sivridis E, Koukourakis MI, Zois CE, Ledaki I, Ferguson DJ, Harris AL, et al. LC3A-positive light microscopy detected patterns of autophagy and prognosis in operable breast carcinomas. Am J Pathol. 2010;176:2477–89. doi: 10.2353/ajpath.2010.090049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Altieri DC, Stein GS, Lian JB, Languino LR. TRAP-1, the mitochondrial Hsp90. Biochim Biophys Acta. 2012;1823:767–73. doi: 10.1016/j.bbamcr.2011.08.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Kang BH. TRAP1 regulation of mitochondrial life or death decision in cancer cells and mitochondria-targeted TRAP1 inhibitors. BMB Rep. 2012;45:1–6. doi: 10.5483/bmbrep.2012.45.1.1. [DOI] [PubMed] [Google Scholar]
- 35.Matassa DS, Amoroso MR, Maddalena F, Landriscina M, Esposito F. New insights into TRAP1 pathway. Am J Cancer Res. 2012;2:235–48. [PMC free article] [PubMed] [Google Scholar]
- 36.Hua G, Zhang Q, Fan Z. Heat shock protein 75 (TRAP1) antagonizes reactive oxygen species generation and protects cells from granzyme M-mediated apoptosis. J Biol Chem. 2007;282:20553–60. doi: 10.1074/jbc.M703196200. [DOI] [PubMed] [Google Scholar]
- 37.Xiang F, Huang YS, Shi XH, Zhang Q. Mitochondrial chaperone tumour necrosis factor receptor-associated protein 1 protects cardiomyocytes from hypoxic injury by regulating mitochondrial permeability transition pore opening. FEBS J. 2010;277:1929–38. doi: 10.1111/j.1742-4658.2010.07615.x. [DOI] [PubMed] [Google Scholar]
- 38.Caino MC, Chae YC, Vaira V, Ferrero S, Nosotti M, Martin NM, et al. Metabolic stress regulates cytoskeletal dynamics and metastasis of cancer cells. J Clin Invest. 2013;123:2907–20. doi: 10.1172/JCI67841. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. doi: 10.1038/ncomms3139. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Sciacovelli M, Guzzo G, Morello V, Frezza C, Zheng L, Nannini N, et al. The Mitochondrial Chaperone TRAP1 Promotes Neoplastic Growth by Inhibiting Succinate Dehydrogenase. Cell Metab. 2013;17:988–99. doi: 10.1016/j.cmet.2013.04.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Ni Chonghaile T, Sarosiek KA, Vo TT, Ryan JA, Tammareddi A, Moore Vdel G, et al. Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science. 2011;334:1129–33. doi: 10.1126/science.1206727. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Sarosiek KA, Ni Chonghaile T, Letai A. Mitochondria: gatekeepers of response to chemotherapy. Trends Cell Biol. 2013;23:612–9. doi: 10.1016/j.tcb.2013.08.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Chien WL, Lee TR, Hung SY, Kang KH, Lee MJ, Fu WM. Impairment of oxidative stress-induced heme oxygenase-1 expression by the defect of Parkinson-related gene of PINK1. J Neurochem. 2011;117:643–53. doi: 10.1111/j.1471-4159.2011.07229.x. [DOI] [PubMed] [Google Scholar]
- 44.Yoshida S, Tsutsumi S, Muhlebach G, Sourbier C, Lee MJ, Lee S, et al. Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis. Proc Natl Acad Sci U S A. 2013;110:E1604–12. doi: 10.1073/pnas.1220659110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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 patients with ovarian cancer -a study of the OVCAD consortium. Mol Cancer. 2012;11:69. doi: 10.1186/1476-4598-11-69. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Gao JY, Song BR, Peng JJ, Lu YM. Correlation between mitochondrial TRAP-1 expression and lymph node metastasis in colorectal cancer. World J Gastroenterol. 2012;18:5965–71. doi: 10.3748/wjg.v18.i41.5965. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.