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Neurotensin expression and outcome of malignant pleural mesothelioma
Biochimie 92 (2010) 164e170
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Research paper
Neurotensin expression and outcome of malignant pleural mesothelioma Marco Alifano a, Mauro Loi a, Sophie Camilleri-Broet b, c, Sandra Dupouy d, Jean François Régnard a, Patricia Forgez d, * a
Département de chirurgie thoracique, Hôtel-Dieu, AP-HP, 1 Place du Parvis Notre-Dame, 75004 Paris, France JE2492-Université Paris-Sud, Hôpital Paul Brousse, 16 av Paul Vaillant Couturier, 94807 Villejuif, France c Cabinet de pathologie Tolbiac, 92 avenue d'Ivry, 75013 Paris, France d UMRS 938 Cdr Saint-Antoine, Pav. R Koulrilsky, Hôpital Saint-Antoine, 184 rue du Fbg Saint-Antoine 75571 Paris cedex 12, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2009 Accepted 13 November 2009 Available online 20 November 2009
Malignant pleural mesothelioma is a frequently fatal disease and the impact of available treatments is globally poor. Identification of new prognostic factors would help in the understanding of disease progression and, possibly, patient management. Here, we evaluate the prognostic impact of the neurotensin (NTS) and its cognate receptor (NTSR1) known for mediating cellular proliferation, survival, invasiveness, and mobility. We studied a series of 52 consecutive patients with epithelioid malignant mesothelioma undergoing management with curative intent, by immunohistochemistry for the expression of NTS and NTSR1. Specimens were scored as 0, 1, or 2 for less than 10%, between 10 and 50%, or more than 50% of NTS positive staining in tumor cells, respectively. Immunohistochemistry revealed that NTS and NTSR1 expression was found in 71.1% and 90.4% of malignant mesotheliomas, respectively. Using univariate analysis, expression of NTS was significantly (p ¼ 0.015) related with a poor prognosis, with median survivals of 11.0 months, 18.4 months, and 29.8 months in patients showing expression scored as 2, 1, and 0, respectively. Multivariate analysis showed that expression of NTS (p ¼ 0.007) and non-surgical therapy (p ¼ 0.004) were independent predictors of poor prognosis. In order to evaluate the role of NTS/NTSR1 complex in mesothelioma progression, in vitro cell invasion assays and wound healing were performed on the mesothelioma cell line, MSTO-211H, and showed that inhibition of the NTS system resulted in a significant reduction of both migration and collagen invasion of mesothelioma cells. The expression of NTS is identified as a prognostic marker in patients with malignant pleural mesothelioma (Patent EP 08305971.7). Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Malignant pleural mesothelioma Neurotensin Prognosis biomarker Cancer progression Cellular migration
1. Introduction Malignant pleural mesothelioma (MPM) is a relatively infrequent disease, whose incidence is estimated in western countries between less than 1 case/million/year among persons non-exposed to asbestos and 100 cases/million/year among individuals professionally exposed to asbestos [1,2]. The incidence of disease is expected to continue to increase in the next years, with a peak between 2015 and 2020 [3]. Prognosis for MPM is poor with the median survival in a comprehensive unselected population being 8.9 months [4]. Reports from referral institutions concerning patients participating in clinical trials showed a slightly better outcome [5e7]. A randomized study comparing mono-chemotherapy using cisplatin, or the association cisplatinepemetrexed, showed a median survival
* Corresponding author. Tel.: þ33 1 49 28 46 74; fax: þ33 1 49 28 66 83. E-mail address: [email protected] (P. Forgez). 0300-9084/$ e see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.11.004
of 9.3 months and 12.1 months in the two arms, respectively [5]. The later drug association is now considered as the standard of care, although a low rate of long term survival is observed [6]. More recent treatment strategy, based on the association of induction chemotherapy and radical surgery by extrapleural pneumonectomy, resulted in an improved survival: median survival of more than 20 months and 5-year survival rates around 40% have been reported in patients with epithelioid mesothelioma, complete (R0) resection, and no mediastinal node involvement [8e12]. Unfortunately, only a minority of patients with MPM will benefit from this aggressive therapeutic strategy, because the advanced disease state, the histological type, the advanced age, and the co-morbidities prevent its used [8e12]. Of the neuropeptideereceptor complexes which are deregulated during the neoplastic process, the 13 amino acid neurotensin (NTS) and its cognate high affinity neurotensin receptor 1 (NTSR1) is one. NTS regulates several biological processes, such as intestinal motility, secretion, vascular smooth muscle activity, and intestinal epithelial cell proliferation, both physiologically [13] and in
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response to acute [14] or chronic injuries [15]. NTS is associated with a number of deleterious functions promoting cancer progression through a number of oncogenic pathways and transforming functions [13]. NTS and NTSR1 expression is found in several human epithelial tumors, such as colon, pancreas, prostate, and breast cancer, as well as in a wide variety of cancer cell lines [13,16]. Evidence is now accumulating showing that activation of the neurotensin system results in cancer progression and worse outcome in both breast, and head and neck squamous cell carcinomas [17,18]. With respect to mesothelioma, transcriptome analysis of the human-derived mesothelioma cell line, MSTO-211H, showed the overexpression of NTS as compared to the nonmalignant transformed mesothelial cells, Met-5A [19]. In this paper we studied the expression of both NTS and NTSR1 in a series of consecutive patients treated for malignant pleural mesothelioma in a curative intent, and evaluated the possible impact on prognosis of the expression of these molecules. Furthermore, using mesothelioma cell line, we demonstrated the effects of the inhibition of the neurotensinergic system on cellular migration and collagen invasion.
serum at room temperature (RT) for 30 min. NTS immunoreactivity was conducted using rabbit antibody directed against NTS (1/500) (NA1230 Biomol, USA) for 2 h at RT in a humidified chamber for tumor. NTSR1 immunoreactivity was detected using a goat polyclonal antibody directed against the human carboxy terminus of the receptor (1:100) (C-20 Santa Cruz USA). All slides were rinsed three times with TBS; sections were incubated with biotinylated secondary antibody (1:200) (Vector, USA), for 30 min at RT. The antigeneantibody complex was revealed with avidinebiotine peroxidase complex, for 30 min, according to the manufacturer's instructions for the Vectastain ABC Kit (Vector, USA). Staining was done for 5 min with diamino-benzidine tetrahydrochloride (DAB) (Sigma, USA). All slides were counterstained with hematoxylin. All specimens were scored by a pathologist (SCB), unaware of treatment modalities and states at follow-up as follows: 0: positive staining <10% of tumor cells; 1: positive staining involving >10% and less than 50% of tumor cells; 2: strong positive staining involving more than 50% of tumor cells.
2. Methods
The human mesothelioma cell line, MSTO-211H, was selected because of its intense proliferative activity and its aggressive profile (ATCC CRL-2081, 211H). MST0-211H are derived from a biphasic mesothelioma: xenograft in nude mice originate tumors with predominance of epithelioid cells and few fibrous cells [21]. Xenograft in nude mice of MST0-211H cell lines results in tumors leading ultimately to animal death [22]. In the present study, the MSTO-211H cells were grown in RPMI1640 medium (Invitrogen, USA) supplemented with 10% fetal calf serum (PAAÔ) and 2 mM glutamine (GibcoÒ). Cells were stored at 37 in 5% CO2 atmosphere.
2.1. Patients Clinical files of patients treated at the Thoracic Surgery Department of Hôtel-Dieu Hospital, Paris, France, between 2002 and 2006 for malignant pleural mesothelioma were retrospectively reviewed. Among these patients, those responding to the following criteria were selected: 1) histological diagnosis of epithelioid malignant mesothelioma on samples obtained at video-assisted thoracoscopy; 2) clinical staging of T1-T3 N0-1 M0 disease; 3) subsequent treatment by either chemotherapy alone (associating Cisplatin and Pemetrexed) or by induction chemotherapy followed by extrapleural pneumonectomy; 4) no death related to the treatment (chemotherapy or surgery); 5) no co-existing malignancy (i.e. other than mesothelioma) diagnosed less than three years before diagnosis of mesothelioma, and, in case of previous malignancy, no evidence of disease recurrence. In almost all cases talc poudrage had been performed during the same operative time as videoassisted thoracoscopy. Also, 26 patients with recurrent idiopathic spontaneous pneumothorax treated by video-assisted thoracoscopy were analyzed as controls. Post-operative treatments and follow-up were carried out under the care of the referring physician, so no uniform protocol was employed. All the patients treated by extrapleural pneumonectomy received post-operative radiotherapy, but dose and schedule were variable. With respect to follow-up, in almost all the instances, patients underwent physical examination, laboratory investigations and thoracoabdomical CT scan every four months in the first three years after completion of treatment with curative intent, and every six months thereafter. Occurrence of suspected or proved recurrence obviously induced to changes in scheduled examinations. 2.2. Tissue specimens and immunohistochemistry For all cases, histologic slides of tumors were selected from the standard H&E staining and adjacent sections of formalin-fixed paraffin-embedded material were obtained for immunohistochemistry. NTS and NTSR1 immunostaining were carried out on 4 mm thick deparaffinized sections, using the avidinebiotineperoxidase complex method, as previously described [20]. Briefly, after inhibition of endogenous peroxidases with 3% hydrogen peroxide, slides were washed in TBS and incubated with 10% normal rabbit
2.3. Culture procedures
2.4. RNA extraction and RT-PCR The protocols for total RNA extraction, reverse-transcription reaction (RT), and PCR are documented in Souaze et al. [23]. RT was performed on 1 mg of total RNA using a specific NTSR1 primer (50 GCTGACGTAGAAGAG-30 ) or 50 pmol of oligo dT and oligo dN. The PCR amplification was performed on a 1:5 v/v of the RT reaction using 25 pmol of each primer 50 -CGTGGAGCTGTACAACTTCA-30 and 50 -CAGCCAGCAGACCACAAAGG-30 for NTSR1, and 50 -AAGCACATG TTCCCTCTT-30 and 50 -CATACAGCTGCCGTTTCAGA-30 for NTS, and 1 unit of Taq polymerase (ABgeneÔ, France). The amplification profile consisted of denaturation at 94 C for 30 s, annealing at 57 C for 45 s, and extension at 72 C for 45 s. The PCR cycle were preceded by denaturation at 95 C for 15 min and were followed by a final extension at 72 C for 7 min. Amplification was performed in a DNA thermal cycler 9700 (Perkin Elmer Applied Biosystems, Courtaboeuf, France). 2.5. Cellular growth Cellular growth was performed using 6 well–culture dishes. 15 104 MSTO-211H cells were seeded, after 24 h cells were washed twice with PBS, and complete media containing either 106 M SR48692, a NTSR1 antagonist, or 104 M de DMSO was added to the cells. After 24, 48, and 72 h of growth, cells were trypsinized and counted with a cells coulter (Z1 CoulterÒ Particle Counter, Beckman CoulterÔ). 2.6. Wound healing assay In order to evaluate the impact of the neurotensinergic system on cell migration, a wound healing assay was performed. 1.75 106
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MSTO-211H cells were plated per well in 6 well dishes containing the medium in order to reach confluence. After 24 h cells were serum-starved and scarification was performed in a Moscona buffer with a 20 ml pipette tip. Cells were maintained in culture with low serum medium (0.5% FCS) and treated with 106 M SR48692. Control cells were exposed or not to the SR48692 solvent, DMSO. Pictures were taken at the beginning and 24 and 30 h later with a photonic microscope. The “remaining gap” was calculated as percentage between wound size at 30 h and initial wound size.
cells were seeded in the inner chamber. Cells were treated with 5 106 M of SR48692 or with 1:50 solution of anti-NTS rabbit antibody (NA1230, Tebu-Bio). Cells were placed for 48 h at 37 C in 5% CO2 atmosphere. After incubation, non-invasive cells on the upper surface of the collagen matrix were wiped off with cotton swabs. Filter membranes were fixed with 5% glutaraldehyde and remaining cells stained with 0.5% toluidine blue. Quantitation of invasive cells was carried out at the 400 magnification by using the software CELINA.
2.7. Collagen invasion assay
2.8. Statistical analysis
Collagen invasion assay was performed by using cell-culture inserts composed of 8 mm porous polycarbonate membrane, placed as inner chamber in 24 well-tissue-culture dishes. This membrane was coated with 40 ml of 2 mg/ml type I collagen diluted in RPMI1640 medium (Collagen type I, rat tail, Upstate). The bottom chamber was filled with 500 ml RPMI-1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine. 103 of MSTO-211H
Survival rates were calculated by the KaplaneMeier estimates and comparison of survival curves were performed with the logrank test. All the available variables possibly influencing survival (sex, age, history of previous neoplasm, asbestos exposure, >5% weight loss, pre-operative CRP levels, duration of symptoms before surgery, surgical treatment, expression of NTS and NTS-R1) were used to carry out a multivariate analysis by using the Cox model.
Fig. 1. NTS (left) and NTSR1 (right) expression in patients with malignant pleural mesothelioma (A), or with idiopathic spontaneous pneumothorax (B). (A) Examples of immunohistochemistry of three different patients are shown. For patient 1 and 2 consecutive slides were used. Original magnifications are noted on the figure. Inset shows computerized enlargement of a region of interest, the black squares localize the image with a black contour. Positivity was scored by an anatomopathologist (Dr S Camilleri-Broët).
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TNM parameters were not included in the analysis because of the excessive low number of subject in each category. Statistical significance was accepted at p < 0.05. The biochemical data were compared by Student's t test or of variances analysis followed by Neuman-Keuls test. 2.9. Ethics Informed consent was obtained by all the patients or relatives (in case of deceased patients) and investigations were conducted according to Declaration of Helsinki principles. The study was carried out according to the French laws on biomedical research. 3. Results Fifty-two consecutive patients responding to the inclusion criteria were considered for the study. There were 34 men and 18 women; mean age was 66 10.5 years. Ten patients had a history of previously treated extrathoracic malignancy, 54% had a history of tobacco smoke, and 65% of work exposure to asbestos. 44% of patients had unintentional loss of body weight >5% in the last three months; whereas 34% had a pre-operative c-reactive protein (CRP), greater than 50 mg/dl. All these patients had diagnostic videoassisted thoracoscopy which was associated to talc poudrage in 50 cases. Chemotherapy by CisplatinePemetrexed was administered in all the cases, which was used for induction purposes in 13 cases and as definitive treatment in the remaining patients. Extrapleural pneumonectomy was carried out in all the 13 patients for whom it was initially planned. 3.1. Expression of NTS and NTSR1 in malignant pleural mesothelioma: immunohistochemistry Expression of NTS was found in 37/52 cases (71.1%) and was scored 1 and 2 in 20 and 17 cases, respectively. In most of the positive cases, labeling was very intense and detected throughout the entire cytosol. For the most part, the staining was homogeneous with nearly all cancer cells positives, whereas the tumor stroma was clearly negative. A typical image of the observed NTS immunostaining is shown in Fig. 1A, right panel. Expression of NTSR1 was found in 47/52 cases (90.4%) and scored as 1 and 2 in 35 and 12 cases, respectively. NTSR1 staining of cancer cells from patients with MPM was granular, mainly restricted to the cytoplasm, and rarely detected at the cell surface, as shown Fig. 1A, left panel. No correlation was found between NTS or NTSR1 expression and the available clinical and pathological parameters other than survival (see below). In pleura of patients treated for idiopathic spontaneous pneumothorax, expression of NTS and NTSR1 was observed in 8/26 (31%) and 20/26 (77%) cases, respectively, whereas no expression of either NTS or NTSR1 was observed in the pulmonary parenchyma of apical resection specimens. In case of positivity of staining of either NTS or NTSR1, it was always scored at 1. Of note, NTSR1 staining in pleura of patients with pneumothorax was in almost all the cases restricted to the cell membrane (Fig. 1B and inset magnification), in contrast to the staining pattern in MPM. 3.2. Survival At the completion of the study in July 2008, 14 were alive and 38 patients had died, with a mean follow-up of 34.8 months. Expression of NTSR1, as assessed by immunohistochemistry, had no impact on survival in patients with MPM (Fig. 2A). On the other hand, the expression of NTS was associated with a significantly (p ¼ 0.015) worse prognosis: median survival was 29.8
Fig. 2. NTSR1 and NTS expression in pleural mesothelioma and global survival at fiveyear duration. Kaplan-Maier analysis and comparison made by the log-rank test was performed (A) on groups with positive (þ) or negative () NTSR1 expression; (B) on groups with absence (), moderate (þ) or strong (þþ) expression for NTS
months, 18.4 months, and 11 months in patients having NTS expression scored as 0, 1, and 2, respectively. Five-year survival was 33.3% (95% C.I. 15.2e58.3 months) in patients with no NTS expression as compared to 0% in case of positive staining scored at 2 (Fig. 2B). With respect to other clinical parameters possibly influencing survival, no difference in terms of survival was observed using univariate analysis according to sex, age or asbestos exposure. High levels of CRP (>50 mg/dl) were associated with a strong trend toward poor prognosis (p ¼ 0.061). On the other hand, patients treated by extrapleural pneumonectomy showed a trend (p ¼ 0.075) toward better prognosis as compared to patients not receiving this surgical treatment (5-year survival rates of 42.3% [95% C.I. 19.8e68.6%] vs 11.0% [95% C.I. 4.0e27.1%], respectively). Using multivariate analysis only expression of NTS (p ¼ 0.007) and a non-surgical treatment (p ¼ 0.04) negatively affected the outcome. 3.3. Expression of the NTS and NTSR1 in MSTO-211H cell line Expression of both NTS and NTSR1 in human malignant mesothelioma MSTO-211 has been investigated in the present study. Two specific PCR amplicons with a size of 210 bp and 434 bp, corresponding respectively to NTS and NTSR1, were detected (Fig. 3A lanes 2 and 4). Correspondence was verified by comparison to the positive control cell line, LNM 35 (a non-small cell lung cancer line), known to express the peptide and the receptor [24] (Fig. 3A lanes 1 and 3). 3.4. Cellular effects of NTS/NTSR1 complex inhibition As shown in Fig. 3B, inhibition of neurotensinergic system by the presence, in the culture media, of a specific NTSR1 antagonist, SR48692, had no effect on cell growth. However, MSTO-211H
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Fig. 3. Effect of NTSR1 antagonist on MSTO-211H cell growth and invasion. A) NTS and NTSR1 transcript analysis. RNA from LNM 35 (lanes 1 and 3) and MSTO-211H (lanes 2 and 4) were reverse-transcribed and PCR was performed as described in the experimental procedures. B) Effect of NTSR1 antagonist on MSTO-211H cellular growth. Cells were treated with 106 M SR48692 or with 104 M DMSO, cells were counted after 24, 48, and 72 h of growth. Results represent the percentage of cells for each time point, compared to the number of cells initially seeded in the culture dishes. C) Effect of NTSR1 blockade on invasive activity of MSTO-21H cells. NTSR1 activity was blocked either with 5 106 M SR48692 or 1/50 dilution NTS antibody. Results represent the percentage of invasive cells compared to the number of cells seeded on the type I collagen coated inner membrane. Results represent the mean SEM from three independent experiments for cellular growth and invasion assay. **, P < 0.01 between DMSO and SR48692 treated cells; £££, P < 0.001 between control and NTS AB treated cells.
invasive ability is significantly decreased when SR48692 or blocking NTS antibody is present in the culture media, only 4% and 5% of cells invaded the type I collagen, respectively, as compared to 13% (untreated cells) and 11% (supplemented with DMSO) in the control groups (Fig. 3C). The wound healing assay showed that residual gap at 24 and 30 h was significantly larger (48% of original gap) when cells are treated with SR48692 as compared to control treatments (27% and 30% of original gap in untreated cells or in the presence of DMSO, respectively; Fig. 4A and B). The wound closure process after scarification of the cell layer, and the cellular invasion activity are, therefore, affected by selective pharmacological blockade of NTSR1.
Fig. 4. Effect of NTSR1 blockade on cellular migration. Wound repair process was studied on MSTO-211H in the presence or absence of NTSR1 antagonist, SR48692. A) Example of wound healing assay: initial gap (T0) and remaining gap (T30 h) of cells treated or not (C) with SR48692 or in the presence of SR48692 solvent (DMSO) is shown. The black marks allow relocating the wound after the first measure. B) Percentage of remaining gap was calculated for control cells, or DMSO or SR48692 treated cells. Remaining gap is the distance of the open wound after 24 or 30 h compared to the initial wound. Results represent the mean SEM from three independent experiments. ***, P < 0.001 between DMSO and SR48692 treated cells.
4. Discussion In the present study we demonstrate that strong NTS expression is associated with poor long term outcome in patients with epithelioid malignant pleural mesothelioma. This finding was confirmed by multivariate analysis which showed that only the
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expression of NTS and the absence of surgery with curative intent (extrapleural pneumonectomy) were independent predictors of poor 5-year survival. Our patient population included only epithelioid mesotheliomas (the more frequent histologic subtype) because in our Institution (as well as in many other ones) aggressive treatments (including extrapleural pneumonectomy) are offered only to patients with this histologic type. This choice allowed to analyze a homogeneous patient population. NTS staining was found in 61% of the mesothelioma samples, but less frequently (30% of cases) and with a slighter intensity in normal pleura specimens. The percentage of specimen expressing NTSR1 was high in both normal pleura and mesothelioma, however the expression pattern was different, as a thin membranous staining was generally observed in normal tissue, whereas a strong intracellular staining was seen in mesothelioma, suggesting an active endocytosis process of the receptor in the latter. The NTS/NTSR1 system is likely quiescent in normal tissue, as opposed to its sustained activation in mesothelioma. Our findings support the case for the participation of NTS signalling pathway in the mesothelioma disease progression. In the clinical evolution of mesothelioma local growth and cellular migration inside the pleural cavity and invasion of the neighboring structure represent characteristic features, whereas distant metastasis often occur relatively late in the evolution of the disease. We addressed potential role of NTS in these processes, using a cellular model expressing endogenously NTS and NTSR1, the MSTO-211H cell line. NTS/NTSR1 blockade by a specific NTSR1 antagonist or NTS blocking antisera strongly reduced both invasion and cellular mobility activity. It was recently shown that NTSR1 activation elicits IKB-alpha degradation and NF-KB activation with subsequent expression and release of IL-8 [25]. IL-8 is a known mediator in the development of pleural effusion in mesothelioma; as well as an autocrine growth factor for malignant pleural mesothelioma cells [26]. Thus, NTS induced chemokine expression and cell migration provides a strong hypothetic role for NTS in the pathogenesis of clinical manifestations of mesothelioma. The present availability of useful prognostic factors in mesothelioma is made difficult due to the extreme heterogeneousness of patient populations, in terms of histological typing, disease staging, and type of treatments. For these reasons, we focused our approach on studying patients whose disease was accessible to a treatment with possibly curative intent. In our series, overall 5-year survival was 19%, and the relative figure were 42.3% and 11% in patients receiving, or not, extrapleural pneumonectomy as a part of their management. All these figures were in agreement with currently available data on patients receiving a similar management. In this selected patient population, we found that only high pre-operative CRP levels (an already known prognostic marker), non-surgical treatment, and high expression of NTS negatively affected outcome. The statistical significance was largely attained for NTS, whereas p values were close to significance for CRP and extrapleural pneumonectomy treatment and reached significance when employing multivariate analysis with respect to NTS expression and treatment by extrapleural pneumonectomy. Amongst patients with high expression of NTS (scored ¼ 2) no survivor was observed beyond 22 months after histological diagnosis. Thus, the detection of NTS expression can help identify of patients requiring stricter follow-up and/or an even more aggressive treatment. Our clinical and experimental data suggest that the NTS/ NTSR1 complex is not only a potential marker of negative prognosis, but also a putative mediator of disease progression. Thus it should be a good candidate for the development of
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specifically targeted drugs, to be used together with currently available treatments. Acknowledgements The authors wish to express many thanks to Dr. Neil Insdorf for his help in the writing of the manuscript. This work has been supported by INSERM and Grants from ARC (Association pour la Recherche sur le Cancer) # 3543, and 3905, and LNC (Ligue national contre le cancer) # 07/75-85 France, GEFLUC 1/184 and MERLION (5-07-06). Sandra Dupouy was supported by the “Ligue nationale contre le cancer”. References [1] [2]
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Research paper
Neurotensin expression and outcome of malignant pleural mesothelioma Marco Alifano a, Mauro Loi a, Sophie Camilleri-Broet b, c, Sandra Dupouy d, Jean François Régnard a, Patricia Forgez d, * a
Département de chirurgie thoracique, Hôtel-Dieu, AP-HP, 1 Place du Parvis Notre-Dame, 75004 Paris, France JE2492-Université Paris-Sud, Hôpital Paul Brousse, 16 av Paul Vaillant Couturier, 94807 Villejuif, France c Cabinet de pathologie Tolbiac, 92 avenue d'Ivry, 75013 Paris, France d UMRS 938 Cdr Saint-Antoine, Pav. R Koulrilsky, Hôpital Saint-Antoine, 184 rue du Fbg Saint-Antoine 75571 Paris cedex 12, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2009 Accepted 13 November 2009 Available online 20 November 2009
Malignant pleural mesothelioma is a frequently fatal disease and the impact of available treatments is globally poor. Identification of new prognostic factors would help in the understanding of disease progression and, possibly, patient management. Here, we evaluate the prognostic impact of the neurotensin (NTS) and its cognate receptor (NTSR1) known for mediating cellular proliferation, survival, invasiveness, and mobility. We studied a series of 52 consecutive patients with epithelioid malignant mesothelioma undergoing management with curative intent, by immunohistochemistry for the expression of NTS and NTSR1. Specimens were scored as 0, 1, or 2 for less than 10%, between 10 and 50%, or more than 50% of NTS positive staining in tumor cells, respectively. Immunohistochemistry revealed that NTS and NTSR1 expression was found in 71.1% and 90.4% of malignant mesotheliomas, respectively. Using univariate analysis, expression of NTS was significantly (p ¼ 0.015) related with a poor prognosis, with median survivals of 11.0 months, 18.4 months, and 29.8 months in patients showing expression scored as 2, 1, and 0, respectively. Multivariate analysis showed that expression of NTS (p ¼ 0.007) and non-surgical therapy (p ¼ 0.004) were independent predictors of poor prognosis. In order to evaluate the role of NTS/NTSR1 complex in mesothelioma progression, in vitro cell invasion assays and wound healing were performed on the mesothelioma cell line, MSTO-211H, and showed that inhibition of the NTS system resulted in a significant reduction of both migration and collagen invasion of mesothelioma cells. The expression of NTS is identified as a prognostic marker in patients with malignant pleural mesothelioma (Patent EP 08305971.7). Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Malignant pleural mesothelioma Neurotensin Prognosis biomarker Cancer progression Cellular migration
1. Introduction Malignant pleural mesothelioma (MPM) is a relatively infrequent disease, whose incidence is estimated in western countries between less than 1 case/million/year among persons non-exposed to asbestos and 100 cases/million/year among individuals professionally exposed to asbestos [1,2]. The incidence of disease is expected to continue to increase in the next years, with a peak between 2015 and 2020 [3]. Prognosis for MPM is poor with the median survival in a comprehensive unselected population being 8.9 months [4]. Reports from referral institutions concerning patients participating in clinical trials showed a slightly better outcome [5e7]. A randomized study comparing mono-chemotherapy using cisplatin, or the association cisplatinepemetrexed, showed a median survival
* Corresponding author. Tel.: þ33 1 49 28 46 74; fax: þ33 1 49 28 66 83. E-mail address: [email protected] (P. Forgez). 0300-9084/$ e see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.11.004
of 9.3 months and 12.1 months in the two arms, respectively [5]. The later drug association is now considered as the standard of care, although a low rate of long term survival is observed [6]. More recent treatment strategy, based on the association of induction chemotherapy and radical surgery by extrapleural pneumonectomy, resulted in an improved survival: median survival of more than 20 months and 5-year survival rates around 40% have been reported in patients with epithelioid mesothelioma, complete (R0) resection, and no mediastinal node involvement [8e12]. Unfortunately, only a minority of patients with MPM will benefit from this aggressive therapeutic strategy, because the advanced disease state, the histological type, the advanced age, and the co-morbidities prevent its used [8e12]. Of the neuropeptideereceptor complexes which are deregulated during the neoplastic process, the 13 amino acid neurotensin (NTS) and its cognate high affinity neurotensin receptor 1 (NTSR1) is one. NTS regulates several biological processes, such as intestinal motility, secretion, vascular smooth muscle activity, and intestinal epithelial cell proliferation, both physiologically [13] and in
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response to acute [14] or chronic injuries [15]. NTS is associated with a number of deleterious functions promoting cancer progression through a number of oncogenic pathways and transforming functions [13]. NTS and NTSR1 expression is found in several human epithelial tumors, such as colon, pancreas, prostate, and breast cancer, as well as in a wide variety of cancer cell lines [13,16]. Evidence is now accumulating showing that activation of the neurotensin system results in cancer progression and worse outcome in both breast, and head and neck squamous cell carcinomas [17,18]. With respect to mesothelioma, transcriptome analysis of the human-derived mesothelioma cell line, MSTO-211H, showed the overexpression of NTS as compared to the nonmalignant transformed mesothelial cells, Met-5A [19]. In this paper we studied the expression of both NTS and NTSR1 in a series of consecutive patients treated for malignant pleural mesothelioma in a curative intent, and evaluated the possible impact on prognosis of the expression of these molecules. Furthermore, using mesothelioma cell line, we demonstrated the effects of the inhibition of the neurotensinergic system on cellular migration and collagen invasion.
serum at room temperature (RT) for 30 min. NTS immunoreactivity was conducted using rabbit antibody directed against NTS (1/500) (NA1230 Biomol, USA) for 2 h at RT in a humidified chamber for tumor. NTSR1 immunoreactivity was detected using a goat polyclonal antibody directed against the human carboxy terminus of the receptor (1:100) (C-20 Santa Cruz USA). All slides were rinsed three times with TBS; sections were incubated with biotinylated secondary antibody (1:200) (Vector, USA), for 30 min at RT. The antigeneantibody complex was revealed with avidinebiotine peroxidase complex, for 30 min, according to the manufacturer's instructions for the Vectastain ABC Kit (Vector, USA). Staining was done for 5 min with diamino-benzidine tetrahydrochloride (DAB) (Sigma, USA). All slides were counterstained with hematoxylin. All specimens were scored by a pathologist (SCB), unaware of treatment modalities and states at follow-up as follows: 0: positive staining <10% of tumor cells; 1: positive staining involving >10% and less than 50% of tumor cells; 2: strong positive staining involving more than 50% of tumor cells.
2. Methods
The human mesothelioma cell line, MSTO-211H, was selected because of its intense proliferative activity and its aggressive profile (ATCC CRL-2081, 211H). MST0-211H are derived from a biphasic mesothelioma: xenograft in nude mice originate tumors with predominance of epithelioid cells and few fibrous cells [21]. Xenograft in nude mice of MST0-211H cell lines results in tumors leading ultimately to animal death [22]. In the present study, the MSTO-211H cells were grown in RPMI1640 medium (Invitrogen, USA) supplemented with 10% fetal calf serum (PAAÔ) and 2 mM glutamine (GibcoÒ). Cells were stored at 37 in 5% CO2 atmosphere.
2.1. Patients Clinical files of patients treated at the Thoracic Surgery Department of Hôtel-Dieu Hospital, Paris, France, between 2002 and 2006 for malignant pleural mesothelioma were retrospectively reviewed. Among these patients, those responding to the following criteria were selected: 1) histological diagnosis of epithelioid malignant mesothelioma on samples obtained at video-assisted thoracoscopy; 2) clinical staging of T1-T3 N0-1 M0 disease; 3) subsequent treatment by either chemotherapy alone (associating Cisplatin and Pemetrexed) or by induction chemotherapy followed by extrapleural pneumonectomy; 4) no death related to the treatment (chemotherapy or surgery); 5) no co-existing malignancy (i.e. other than mesothelioma) diagnosed less than three years before diagnosis of mesothelioma, and, in case of previous malignancy, no evidence of disease recurrence. In almost all cases talc poudrage had been performed during the same operative time as videoassisted thoracoscopy. Also, 26 patients with recurrent idiopathic spontaneous pneumothorax treated by video-assisted thoracoscopy were analyzed as controls. Post-operative treatments and follow-up were carried out under the care of the referring physician, so no uniform protocol was employed. All the patients treated by extrapleural pneumonectomy received post-operative radiotherapy, but dose and schedule were variable. With respect to follow-up, in almost all the instances, patients underwent physical examination, laboratory investigations and thoracoabdomical CT scan every four months in the first three years after completion of treatment with curative intent, and every six months thereafter. Occurrence of suspected or proved recurrence obviously induced to changes in scheduled examinations. 2.2. Tissue specimens and immunohistochemistry For all cases, histologic slides of tumors were selected from the standard H&E staining and adjacent sections of formalin-fixed paraffin-embedded material were obtained for immunohistochemistry. NTS and NTSR1 immunostaining were carried out on 4 mm thick deparaffinized sections, using the avidinebiotineperoxidase complex method, as previously described [20]. Briefly, after inhibition of endogenous peroxidases with 3% hydrogen peroxide, slides were washed in TBS and incubated with 10% normal rabbit
2.3. Culture procedures
2.4. RNA extraction and RT-PCR The protocols for total RNA extraction, reverse-transcription reaction (RT), and PCR are documented in Souaze et al. [23]. RT was performed on 1 mg of total RNA using a specific NTSR1 primer (50 GCTGACGTAGAAGAG-30 ) or 50 pmol of oligo dT and oligo dN. The PCR amplification was performed on a 1:5 v/v of the RT reaction using 25 pmol of each primer 50 -CGTGGAGCTGTACAACTTCA-30 and 50 -CAGCCAGCAGACCACAAAGG-30 for NTSR1, and 50 -AAGCACATG TTCCCTCTT-30 and 50 -CATACAGCTGCCGTTTCAGA-30 for NTS, and 1 unit of Taq polymerase (ABgeneÔ, France). The amplification profile consisted of denaturation at 94 C for 30 s, annealing at 57 C for 45 s, and extension at 72 C for 45 s. The PCR cycle were preceded by denaturation at 95 C for 15 min and were followed by a final extension at 72 C for 7 min. Amplification was performed in a DNA thermal cycler 9700 (Perkin Elmer Applied Biosystems, Courtaboeuf, France). 2.5. Cellular growth Cellular growth was performed using 6 well–culture dishes. 15 104 MSTO-211H cells were seeded, after 24 h cells were washed twice with PBS, and complete media containing either 106 M SR48692, a NTSR1 antagonist, or 104 M de DMSO was added to the cells. After 24, 48, and 72 h of growth, cells were trypsinized and counted with a cells coulter (Z1 CoulterÒ Particle Counter, Beckman CoulterÔ). 2.6. Wound healing assay In order to evaluate the impact of the neurotensinergic system on cell migration, a wound healing assay was performed. 1.75 106
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MSTO-211H cells were plated per well in 6 well dishes containing the medium in order to reach confluence. After 24 h cells were serum-starved and scarification was performed in a Moscona buffer with a 20 ml pipette tip. Cells were maintained in culture with low serum medium (0.5% FCS) and treated with 106 M SR48692. Control cells were exposed or not to the SR48692 solvent, DMSO. Pictures were taken at the beginning and 24 and 30 h later with a photonic microscope. The “remaining gap” was calculated as percentage between wound size at 30 h and initial wound size.
cells were seeded in the inner chamber. Cells were treated with 5 106 M of SR48692 or with 1:50 solution of anti-NTS rabbit antibody (NA1230, Tebu-Bio). Cells were placed for 48 h at 37 C in 5% CO2 atmosphere. After incubation, non-invasive cells on the upper surface of the collagen matrix were wiped off with cotton swabs. Filter membranes were fixed with 5% glutaraldehyde and remaining cells stained with 0.5% toluidine blue. Quantitation of invasive cells was carried out at the 400 magnification by using the software CELINA.
2.7. Collagen invasion assay
2.8. Statistical analysis
Collagen invasion assay was performed by using cell-culture inserts composed of 8 mm porous polycarbonate membrane, placed as inner chamber in 24 well-tissue-culture dishes. This membrane was coated with 40 ml of 2 mg/ml type I collagen diluted in RPMI1640 medium (Collagen type I, rat tail, Upstate). The bottom chamber was filled with 500 ml RPMI-1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine. 103 of MSTO-211H
Survival rates were calculated by the KaplaneMeier estimates and comparison of survival curves were performed with the logrank test. All the available variables possibly influencing survival (sex, age, history of previous neoplasm, asbestos exposure, >5% weight loss, pre-operative CRP levels, duration of symptoms before surgery, surgical treatment, expression of NTS and NTS-R1) were used to carry out a multivariate analysis by using the Cox model.
Fig. 1. NTS (left) and NTSR1 (right) expression in patients with malignant pleural mesothelioma (A), or with idiopathic spontaneous pneumothorax (B). (A) Examples of immunohistochemistry of three different patients are shown. For patient 1 and 2 consecutive slides were used. Original magnifications are noted on the figure. Inset shows computerized enlargement of a region of interest, the black squares localize the image with a black contour. Positivity was scored by an anatomopathologist (Dr S Camilleri-Broët).
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TNM parameters were not included in the analysis because of the excessive low number of subject in each category. Statistical significance was accepted at p < 0.05. The biochemical data were compared by Student's t test or of variances analysis followed by Neuman-Keuls test. 2.9. Ethics Informed consent was obtained by all the patients or relatives (in case of deceased patients) and investigations were conducted according to Declaration of Helsinki principles. The study was carried out according to the French laws on biomedical research. 3. Results Fifty-two consecutive patients responding to the inclusion criteria were considered for the study. There were 34 men and 18 women; mean age was 66 10.5 years. Ten patients had a history of previously treated extrathoracic malignancy, 54% had a history of tobacco smoke, and 65% of work exposure to asbestos. 44% of patients had unintentional loss of body weight >5% in the last three months; whereas 34% had a pre-operative c-reactive protein (CRP), greater than 50 mg/dl. All these patients had diagnostic videoassisted thoracoscopy which was associated to talc poudrage in 50 cases. Chemotherapy by CisplatinePemetrexed was administered in all the cases, which was used for induction purposes in 13 cases and as definitive treatment in the remaining patients. Extrapleural pneumonectomy was carried out in all the 13 patients for whom it was initially planned. 3.1. Expression of NTS and NTSR1 in malignant pleural mesothelioma: immunohistochemistry Expression of NTS was found in 37/52 cases (71.1%) and was scored 1 and 2 in 20 and 17 cases, respectively. In most of the positive cases, labeling was very intense and detected throughout the entire cytosol. For the most part, the staining was homogeneous with nearly all cancer cells positives, whereas the tumor stroma was clearly negative. A typical image of the observed NTS immunostaining is shown in Fig. 1A, right panel. Expression of NTSR1 was found in 47/52 cases (90.4%) and scored as 1 and 2 in 35 and 12 cases, respectively. NTSR1 staining of cancer cells from patients with MPM was granular, mainly restricted to the cytoplasm, and rarely detected at the cell surface, as shown Fig. 1A, left panel. No correlation was found between NTS or NTSR1 expression and the available clinical and pathological parameters other than survival (see below). In pleura of patients treated for idiopathic spontaneous pneumothorax, expression of NTS and NTSR1 was observed in 8/26 (31%) and 20/26 (77%) cases, respectively, whereas no expression of either NTS or NTSR1 was observed in the pulmonary parenchyma of apical resection specimens. In case of positivity of staining of either NTS or NTSR1, it was always scored at 1. Of note, NTSR1 staining in pleura of patients with pneumothorax was in almost all the cases restricted to the cell membrane (Fig. 1B and inset magnification), in contrast to the staining pattern in MPM. 3.2. Survival At the completion of the study in July 2008, 14 were alive and 38 patients had died, with a mean follow-up of 34.8 months. Expression of NTSR1, as assessed by immunohistochemistry, had no impact on survival in patients with MPM (Fig. 2A). On the other hand, the expression of NTS was associated with a significantly (p ¼ 0.015) worse prognosis: median survival was 29.8
Fig. 2. NTSR1 and NTS expression in pleural mesothelioma and global survival at fiveyear duration. Kaplan-Maier analysis and comparison made by the log-rank test was performed (A) on groups with positive (þ) or negative () NTSR1 expression; (B) on groups with absence (), moderate (þ) or strong (þþ) expression for NTS
months, 18.4 months, and 11 months in patients having NTS expression scored as 0, 1, and 2, respectively. Five-year survival was 33.3% (95% C.I. 15.2e58.3 months) in patients with no NTS expression as compared to 0% in case of positive staining scored at 2 (Fig. 2B). With respect to other clinical parameters possibly influencing survival, no difference in terms of survival was observed using univariate analysis according to sex, age or asbestos exposure. High levels of CRP (>50 mg/dl) were associated with a strong trend toward poor prognosis (p ¼ 0.061). On the other hand, patients treated by extrapleural pneumonectomy showed a trend (p ¼ 0.075) toward better prognosis as compared to patients not receiving this surgical treatment (5-year survival rates of 42.3% [95% C.I. 19.8e68.6%] vs 11.0% [95% C.I. 4.0e27.1%], respectively). Using multivariate analysis only expression of NTS (p ¼ 0.007) and a non-surgical treatment (p ¼ 0.04) negatively affected the outcome. 3.3. Expression of the NTS and NTSR1 in MSTO-211H cell line Expression of both NTS and NTSR1 in human malignant mesothelioma MSTO-211 has been investigated in the present study. Two specific PCR amplicons with a size of 210 bp and 434 bp, corresponding respectively to NTS and NTSR1, were detected (Fig. 3A lanes 2 and 4). Correspondence was verified by comparison to the positive control cell line, LNM 35 (a non-small cell lung cancer line), known to express the peptide and the receptor [24] (Fig. 3A lanes 1 and 3). 3.4. Cellular effects of NTS/NTSR1 complex inhibition As shown in Fig. 3B, inhibition of neurotensinergic system by the presence, in the culture media, of a specific NTSR1 antagonist, SR48692, had no effect on cell growth. However, MSTO-211H
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Fig. 3. Effect of NTSR1 antagonist on MSTO-211H cell growth and invasion. A) NTS and NTSR1 transcript analysis. RNA from LNM 35 (lanes 1 and 3) and MSTO-211H (lanes 2 and 4) were reverse-transcribed and PCR was performed as described in the experimental procedures. B) Effect of NTSR1 antagonist on MSTO-211H cellular growth. Cells were treated with 106 M SR48692 or with 104 M DMSO, cells were counted after 24, 48, and 72 h of growth. Results represent the percentage of cells for each time point, compared to the number of cells initially seeded in the culture dishes. C) Effect of NTSR1 blockade on invasive activity of MSTO-21H cells. NTSR1 activity was blocked either with 5 106 M SR48692 or 1/50 dilution NTS antibody. Results represent the percentage of invasive cells compared to the number of cells seeded on the type I collagen coated inner membrane. Results represent the mean SEM from three independent experiments for cellular growth and invasion assay. **, P < 0.01 between DMSO and SR48692 treated cells; £££, P < 0.001 between control and NTS AB treated cells.
invasive ability is significantly decreased when SR48692 or blocking NTS antibody is present in the culture media, only 4% and 5% of cells invaded the type I collagen, respectively, as compared to 13% (untreated cells) and 11% (supplemented with DMSO) in the control groups (Fig. 3C). The wound healing assay showed that residual gap at 24 and 30 h was significantly larger (48% of original gap) when cells are treated with SR48692 as compared to control treatments (27% and 30% of original gap in untreated cells or in the presence of DMSO, respectively; Fig. 4A and B). The wound closure process after scarification of the cell layer, and the cellular invasion activity are, therefore, affected by selective pharmacological blockade of NTSR1.
Fig. 4. Effect of NTSR1 blockade on cellular migration. Wound repair process was studied on MSTO-211H in the presence or absence of NTSR1 antagonist, SR48692. A) Example of wound healing assay: initial gap (T0) and remaining gap (T30 h) of cells treated or not (C) with SR48692 or in the presence of SR48692 solvent (DMSO) is shown. The black marks allow relocating the wound after the first measure. B) Percentage of remaining gap was calculated for control cells, or DMSO or SR48692 treated cells. Remaining gap is the distance of the open wound after 24 or 30 h compared to the initial wound. Results represent the mean SEM from three independent experiments. ***, P < 0.001 between DMSO and SR48692 treated cells.
4. Discussion In the present study we demonstrate that strong NTS expression is associated with poor long term outcome in patients with epithelioid malignant pleural mesothelioma. This finding was confirmed by multivariate analysis which showed that only the
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expression of NTS and the absence of surgery with curative intent (extrapleural pneumonectomy) were independent predictors of poor 5-year survival. Our patient population included only epithelioid mesotheliomas (the more frequent histologic subtype) because in our Institution (as well as in many other ones) aggressive treatments (including extrapleural pneumonectomy) are offered only to patients with this histologic type. This choice allowed to analyze a homogeneous patient population. NTS staining was found in 61% of the mesothelioma samples, but less frequently (30% of cases) and with a slighter intensity in normal pleura specimens. The percentage of specimen expressing NTSR1 was high in both normal pleura and mesothelioma, however the expression pattern was different, as a thin membranous staining was generally observed in normal tissue, whereas a strong intracellular staining was seen in mesothelioma, suggesting an active endocytosis process of the receptor in the latter. The NTS/NTSR1 system is likely quiescent in normal tissue, as opposed to its sustained activation in mesothelioma. Our findings support the case for the participation of NTS signalling pathway in the mesothelioma disease progression. In the clinical evolution of mesothelioma local growth and cellular migration inside the pleural cavity and invasion of the neighboring structure represent characteristic features, whereas distant metastasis often occur relatively late in the evolution of the disease. We addressed potential role of NTS in these processes, using a cellular model expressing endogenously NTS and NTSR1, the MSTO-211H cell line. NTS/NTSR1 blockade by a specific NTSR1 antagonist or NTS blocking antisera strongly reduced both invasion and cellular mobility activity. It was recently shown that NTSR1 activation elicits IKB-alpha degradation and NF-KB activation with subsequent expression and release of IL-8 [25]. IL-8 is a known mediator in the development of pleural effusion in mesothelioma; as well as an autocrine growth factor for malignant pleural mesothelioma cells [26]. Thus, NTS induced chemokine expression and cell migration provides a strong hypothetic role for NTS in the pathogenesis of clinical manifestations of mesothelioma. The present availability of useful prognostic factors in mesothelioma is made difficult due to the extreme heterogeneousness of patient populations, in terms of histological typing, disease staging, and type of treatments. For these reasons, we focused our approach on studying patients whose disease was accessible to a treatment with possibly curative intent. In our series, overall 5-year survival was 19%, and the relative figure were 42.3% and 11% in patients receiving, or not, extrapleural pneumonectomy as a part of their management. All these figures were in agreement with currently available data on patients receiving a similar management. In this selected patient population, we found that only high pre-operative CRP levels (an already known prognostic marker), non-surgical treatment, and high expression of NTS negatively affected outcome. The statistical significance was largely attained for NTS, whereas p values were close to significance for CRP and extrapleural pneumonectomy treatment and reached significance when employing multivariate analysis with respect to NTS expression and treatment by extrapleural pneumonectomy. Amongst patients with high expression of NTS (scored ¼ 2) no survivor was observed beyond 22 months after histological diagnosis. Thus, the detection of NTS expression can help identify of patients requiring stricter follow-up and/or an even more aggressive treatment. Our clinical and experimental data suggest that the NTS/ NTSR1 complex is not only a potential marker of negative prognosis, but also a putative mediator of disease progression. Thus it should be a good candidate for the development of
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specifically targeted drugs, to be used together with currently available treatments. Acknowledgements The authors wish to express many thanks to Dr. Neil Insdorf for his help in the writing of the manuscript. This work has been supported by INSERM and Grants from ARC (Association pour la Recherche sur le Cancer) # 3543, and 3905, and LNC (Ligue national contre le cancer) # 07/75-85 France, GEFLUC 1/184 and MERLION (5-07-06). Sandra Dupouy was supported by the “Ligue nationale contre le cancer”. References [1] [2]
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