Brain-derived neurotrophic factor, a new soluble biomarker for malignant pleural mesothelioma involved in angiogenesis
Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer related to asbestos exposure. The discovery of soluble biomarkers with diagnostic/prognostic and/or therapeutic properties would improve therapeutic care of MPM patients. Currently, soluble biomarkers described present weaknesses preventing their use in clinic. This study aimed at evaluating brain-derived neurotrophic factor (BDNF), we previously identified using transcriptomic approach, in MPM. We observed that high BDNF expression, at the mRNA level in tumors or at the protein level in pleural effusions (PE), was a specific hallmark of MPM samples. This protein presented significant but limited diagnostic properties (area under the curve (AUC) = 0.6972, p < 0.0001). Interestingly, high BDNF gene expression and PE concentration were predictive of shorter MPM patient survival (13.0 vs 8.3 months, p < 0.0001, in PE). Finally, BDNF did not affect MPM cell oncogenic properties but was implicated in PE-induced angiogenesis. In conclusion, BDNF appears to be a new interesting biomarker for MPM and could also be a new therapeutic target regarding its implication in angiogenesis.
KeywordsBDNF Mesothelioma Pleural effusions Biomarkers Angiogenesis
Area under the curve
Brain-derived neurotrophic factor
Benign pleural effusion
Cell culture supernatant
Enzyme-linked immunosorbent assay
Fetal calf serum
Human umbilical vein endothelial cell
Lung squamous cell carcinoma
Malignant pleural mesothelioma
Polymerase chain reaction
Receiver operating characteristic
Roswell Park Memorial Institute medium
- RSEM values
RNA-seq by Expectation Maximization values
Soluble mesothelin-related peptide
The Cancer Genome Atlas
Tropomyosin-related kinase receptors B
Vascular endothelial growth factor
Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer related to asbestos exposure. The first line regimen for MPM, consisting of a combination of cisplatin and the anti-metabolite pemetrexed, only increases patient survival by 3 months . The late diagnosis of the disease is partly responsible for the poor outcome in MPM. Thus, the identification of new biomarkers with diagnostic/prognostic and/or therapeutic properties would be useful to improve the therapeutic care of patients and the outcome of the disease. Soluble biomarkers have the advantage of being easily measured in fluid samples without the need to resort to invasive procedures and also to be targetable using antibodies. Previously identified MPM soluble biomarkers, soluble mesothelin-related peptide (SMRP) and fibulin-3, are too limited to be used routinely in clinic and are not identified as therapeutic target . Therefore, the identification of new soluble biomarkers with improved or complementary properties is required.
In a previous study, we identified BDNF, a neurotrophin, as an interesting biomarker for MPM . In this work, we aimed at examining this potential using collections of MPM samples. We also studied the implication of BDNF in MPM pathology.
Results and discussion
BDNF mRNA expression in MPM tumors and prognostic value
BDNF expression and overall survival of patients were related (Fig. 1b and Additional file 4: Table S2). Indeed, patients with high BDNF had a lower survival than patients with low BDNF (15.9 versus 21.1 months, p = 0.0736) and this survival difference is significant at 3 years (p = 0.0401).
These observations were confirmed using TCGA database (Additional file 2: Table S1.2). Expression of BDNF was significantly higher in MPM than in lung squamous carcinoma and lung adenocarcinoma (Fig. 1c). As previously observed, high BDNF was associated with low survival compared to low BDNF (12.4 versus 27.5 months, p < 0.0001) (Fig. 1d and Additional file 4: Table S2). BDNF was already described as overexpressed in several other cancers . In TCGA cohort, we observed that MPM has the highest BDNF expression among 37 tumor types indicating that BDNF gene overexpression is a hallmark of MPM (Fig. 1e and Additional file 5: Table S3). These results were confirmed at the mRNA level and using Immunofluorescence on cancer cell lines and commercial primary mesothelial cells (MC) (Additional file 6: Figure S3A-B).
Expression of BDNF in pleural effusions from patients
These results confirmed a preliminary observation by Duysinx and colleagues performed on only 10 MPM PE  and can be explained, in part, by the ability of MPM cells to produce high level of BDNF (Additional file 6: Figure S3C). This growth factor can also be produced by a large variety of cells  explaining its presence in other PE, but at a lower amount.
Area under the curve (AUC) of BDNF to differentiate MPM from other neoplasia or all PE were similar (AUC = 0.6710 ± 0.04 and AUC = 0.6972 ± 0.038) (Fig. 2c and Additional file 7: Table S4.1). The best specificity and sensitivity for BDNF were ~ 86.05% and ~ 49.51% (Additional file 7: Table S4.2).
The diagnostic value of BDNF (AUC = 0.69) seems slightly lower than the one of SMRP (AUC = 0.76 to 0.87) , the best MPM soluble biomarker to date. However, BDNF is expressed by all subtypes of MPM unlike SMRP which is not expressed by SM . Then, an association of these two biomarkers has a strong potential to improve the sensitivity and the specificity of MPM diagnosis. Comparison of BDNF diagnostic value with fibulin-3 is currently complicated due to heterogeneity in the results obtained with this biomarker .
Prognostic value of BDNF in pleural effusions from patients
In several cancers, BDNF was described as overexpressed in the tumor environment [4, 7] and can be associated with poor survival . Then, we evaluated the prognosis value of BDNF in MPM PE. Interestingly, as in mRNA study, patients with BDNF above median presented a significantly lower survival than the others (8.3 versus 13 months; p = 0.0061) (Fig. 2d and Additional file 4: Table S2). This association between high BDNF and poor survival suggests an implication of this protein in the development of the pathology.
Whereas prognostic value of SMRP remains inconclusive , patients with high BDNF have a shorter survival than patients with low BDNF. In PE, this observation is not related to MPM subtype. Indeed, in this cohort, SM, the most aggressive subtype of mesothelioma, only represent 7% of the cases and therefore cannot be responsible for this result. In PE, these characteristics are similar to the prognostic value of Fibulin-3 .
Evaluation of BDNF on angiogenesis
BDNF was also described as involved on angiogenesis in different cancer types . We thus studied this property by measuring the induction of HUVEC proliferation. First, we showed that MPM PE induced angiogenesis by leading to an increase of HUVEC tube formation and proliferation (Additional file 9: Figure S5A-B). Figure 3c shows that an anti-BDNF blocking antibody (from rabbit, Abcam) reduced significantly by ~ 31% the MPM PE-induced HUVEC proliferation. A detailed analysis of the results led to the segregation of the MPM PE in a sensitive group to BDNF blocking (n = 11) and in a resistant group (n = 3) (Fig. 3d). These results were confirmed using another anti-BDNF blocking antibody (from chicken, Abcam) (Additional file 9: Figure S5C).
These observations demonstrate the strong implication of BDNF in the PE-induced angiogenesis. However, the resistance of some PE to the blocking antibody demonstrates that BDNF is not the only player participating to this process. This is also supported by the observation that the activity of the blocking antibody is not correlated to BDNF concentrations in PE (Additional file 10: Figure S6). Previous works have shown that, in some cancers, BDNF can induce expression of the vascular endothelial growth factor (VEGF), well known to induce angiogenesis, . Thus, we measured VEGF in MPM PE. No evident correlation between BDNF and VEGF was observed (Additional file 11: Figure S7A). However, we did not observe samples with high BDNF and low VEGF. Moreover, in PE with BDNF higher than median value, a positive correlation with VEGF was observed (Additional file 11: Figure S7B). This suggests that VEGF can be dependent of BDNF in some PE. As observed for BDNF, the activity of the blocking antibody was not correlated to VEGF concentrations (Additional file 11: Figure S7C). These results show that VEGF cannot explain anti-angiogenic effect of the BDNF blocking antibody.
Recently, in the MAPS study, it was shown that the combination pemetrexed/cisplatin in association with bevacizumab (anti-VEGF) improves overall survival of MPM patients . This clinical trial demonstrates the interest of targeting angiogenesis in MPM. Regarding our results, this suggests that BDNF could be an interesting target in MPM due to its implication in this process.
Our work identifies BDNF as new interesting MPM biomarker. Moreover, due to its implication in angiogenesis, BDNF could also be a new potential therapeutic target.
The authors want to thank the ‘attachées de recherche clinique’ Megguy Bernard and Régine Valéro for management of sample collection and update of biocollection database, the staff of Laënnec hospital and the MicroPiCell core facility for microscopy experiments.
This work was supported by INSERM, CNRS, the ‘Institut de recherche en santé respiratoire des Pays de la Loire’, the ‘Ligue Contre le Cancer (committees of Morbihan, Sarthe, Vendée et Loire-Atlantique) and ARSMESO44.
Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author. Materials and methods are provided as Additional file 12.
CB, DJ and MG: responsible for study design and execution, data collection, data analysis and manuscript preparation. PS, SMD, ALC, CL, LC, SD, CM, FM, MCC, PH, LPB, and HP: responsible for study execution and data collection. SB and AS: responsible for data analysis and manuscript preparation. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All recruited patients gave signed, informed consent. All the collected samples and the associated clinical information were registered in database (DC-2011-1399 and DC-2013-1963) validated by the French research ministry.
Consent for publication
The authors declare that they have no competing interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 10.Zalcman G, Mazieres J, Margery J, Greillier L, Audigier-Valette C, Moro-Sibilot D, et al. Bevacizumab for newly diagnosed pleural mesothelioma in the mesothelioma Avastin cisplatin Pemetrexed study (MAPS): a randomised, controlled, open-label, phase 3 trial. Lancet. 2016;387:1405–14.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.