Background
Glioblastoma (GBM; World Health Organization grade IV astrocytoma) is the most common malignant brain tumor with an annual incidence of 3.5 cases per 100,000 worldwide [
1]. It is also one of the most lethal human cancers. The median overall survival is 12–15 months with standard treatment [
2], and 3–6 months for patients with recurrent GBM [
3]. Owing to uncertainty in clinical outcome in individual patients, new prognostic markers are needed for GBM patients, especially those with potential to affect patient outcome through druggable targets.
Endothelins are vasoactive peptides that exert their effects through interactions with the G-protein-coupled receptors endothelin receptor A (ETAR) and endothelin receptor B (ETBR). ETAR is expressed mainly in vascular smooth muscle cells and stromal cells, whereas ETBR is expressed mainly in endothelial cells; ETAR mediates vasoconstriction, and ETBR vasodilatation and also stimulates cell proliferation (reviewed in [
4]. Dysregulation of ETBR has been implicated in cardiovascular disease and linked to a congenital disorder, Hirschsprung’s disease (reviewed in [
4]). Moreover, ETBR is overexpressed in vulvar cancer [
5], clear-cell renal cell carcinoma [
6], and esophageal squamous cell carcinoma [
7] and is closely associated with disease progression and poor patient survival [
5‐
7]. Consistent with a crucial role for ETBR in tumorigenesis, some ETBR antagonists may be beneficial in treating melanoma or glioma [
8‐
11].
Overexpression of ETBR in GBM was associated with a poor prognosis in a Chinese population [
12]. Since ethnicity may play a major role in the pathogenesis of gliomas [
13,
14], we investigated whether ETBR overexpression could be detected in patients with GBM and in other cancers outside of China, whether ETBR expression correlates with patient survival (and thus its potential use as a prognostic marker and/or therapeutic target), and whether clinically available endothelin receptor blockers/antagonists have toxic effects on cancer cells in vitro.
Methods
Patient cohort
Formalin-fixed, paraffin-embedded tissue sections were from 25 GBM cases, were selected from our previously studied cohort without prior selection [
15]. Demographic information and clinical data with time to tumor progression (TTP) and overall survival (OS) for all GBM cases are shown in Table
1. Ten normal samples from aging control brains (frontal part of brain from men) median age 57 [50-61 yrs] were from the Department of Pathology, University of Malaya Medical Center (ethical number 896.7). The use of patient materials was approved by the Ethics Committee at the Karolinska Institutet and by the Medical Ethics Committee, University of Malaya Medical Center, Malaysia, and conducted in accordance with the Declaration of Helsinki.
Table 1
Demographic and available clinical information
K7686-2004 | 4 | 5 | 3+ | 73 | M | No | Yes |
K9802-2004 | 5 | 5 | 1+ | 68 | M | Yes | No |
K4448-2004 | 1 | 14 | 1+ | 66 | M | No | Yes |
K12700-2004 | 1 | 5 | 1+ | 64 | F | Yes | No |
K10452-2004 | 7 | 10 | 1+ | 59 | F | Yes | No |
K5126-2004 | 3 | 7 | 1+ | 57 | M | Yes | No |
K11136-2004 | 12 | 20 | 1+ | 56 | M | Yes | No |
K17437-2004 | 16 | 17 | 2+ | 56 | M | Yes | No |
K4840-2004 | 7 | 20 | 1+ | 55 | M | Yes | No |
K16204-2004 | 10 | 11 | 1+ | 54 | M | Yes | No |
K9236-2004 | 12 | 15 | 1+ | 49 | F | Yes | No |
K16178-2004 | 12 | 13 | 1+ | 45 | M | Yes | No |
K3839-2004 | 12 | 14 | 3+ | 28 | M | Yes | No |
K16102-2004 | 48 | 48 | 1+ | 57 | F | Yes | No |
K17407-2004 | 48 | 48 | 1+ | 26 | F | Yes | No |
K10315-2004 | 52 | 52 | 1+ | 29 | M | Yes | No |
K3174-2004 | 15 | 19 | 3+ | 79 | F | Yes | No |
K16595/04 | 17 | 36 | 1+ | 53 | F | Yes | No |
K1716-2005 | 7 | 82 | 1+ | 43 | M | Yes | No |
K3349-2005 | 3 | 3 | 3+ | 79 | F | No | Yes |
K8622-2005 | 9 | 12 | 3+ | 59 | F | Yes | No |
K9731-2005 | 4 | 7 | 3+ | 52 | F | Yes | No |
K15725-2005 | 2 | 4 | 2+ | 54 | M | Yes | No |
K16886-2005 | 4.5 | 14.5 | 2+ | 38 | F | No | Yes |
K17972-2005 | 8.5 | 18 | 1+ | 63 | F | Yes | No |
ETBR immunohistochemistry
Formalin-fixed, paraffin-embedded sections were analyzed by immunohistochemistry as described but with minor modifications [
7]. In brief, the sections were deparaffinized and rehydrated in a graded series of ethanol, and antigen was retrieved with the Decloaking Chamber NxGen (Biocare Medical, Concord, CA, USA) and Antigen Retrieval Citra Plus solution (Biogenex, Emergo Europe, The Hague, The Netherlands) at 110 °C for 15 min. The sections were cooled to room temperature, equilibrated with Tris-buffered saline, pH 7.6, and subjected to a series of blocking steps with protein block (Dako Sweden, Stockholm, Sweden), Fc receptor blocker (Biogenex), and normal horse serum. The sections were then incubated with primary rabbit anti-ETBR (cat. no. E9905; 1:200, Sigma-Aldrich, Stockholm, Sweden) at 4 °C for 16 h, washed three times with Tris-buffered saline, and placed in 3% (
v/v) H
2O
2 in water for 15 min at room temperature to quench endogenous peroxidase activity. After three washings with Tris-buffered saline, the sections were incubated with secondary anti-rabbit antibody conjugated to horseradish peroxidase (ImmPRESS kit, Vector Laboratories, Orton Southgate, Peterborough, UK). Immunoreactivity was revealed with diaminobenzidine (Innovex Biosciences, GENTAUR Europe BVBA, Belgium). The sections were then counterstained with hematoxylin, dried, and mounted with xylene-based mounting medium. Positive staining was graded as low or high as described [
12].
Analysis of data from the cancer genome atlas (TCGA) and the genome expression omnibus (GEO)
To evaluate the ETBR expression profile in GBM patients, we obtained primary and processed gene expression data for TCGA GBM cohort from The Broad Institute TCGA GDAC Firehose (
https://gdac.broadinstitute.org/) using RTCGA (
http://rtcga.github.io/RTCGA). Kaplan-Meier survival curves were plotted using the clinical information submitted for GBM patients in TCGA. GEO datasets related to GBM (GSE2223, GSE7696, GSE16011, GSE10878, GSE46016, GSE15824, GSE31262, GSE42656, and GSE50161) were analyzed for ETBR expression normalized to control. Among selected studies GSE2223 (origin: normal brain samples and glioblastoma), GSE7696 (origin: non-tumoral brain samples and glioblastoma samples with radiotherapy or TMZ/radiotherapy, age 27-70 years), GSE10878 (origin: normal tissue and primary glioblastoma tissue, age: 39-76 years), GSE46016 (origin: neural stem cells and human glioblastoma stem cells, age: 33-71 years), GSE15824 (origin: normal brain tissue & astrocytes and primary glioblastoma tissue, age: 35-70 years), GSE31262 (origin: non-tumoral brain tissue and primary glioblastoma tissue, age:33-71 years), GSE42656 (origin: adult control cerebellum and pediatrics glioblastoma, age:1-82 weeks), and GSE50161 (origin: normal brain samples and primary tumor (glioblastoma), age: 35-70 years) were analyzed. Other types of gliomas such as astrocytoma, oligodendroglioma, medulloblastoma were not considered for the analysis. The data was log2 transformed and then a differential analysis was performed using standard Limma function (R package for the analysis of differential gene expression [
16]). The normalized data further scaled between 0 and 1 using formula Zi = xi - min(x) / max(x) - min(x), where x represent the expression.
Protein–protein interaction network
Information on proteins that interact with ETBR was obtained with a protein neighborhood analysis tool [
17]. The interacting partners were shown with Cytoscape, an online open source software tool to display molecular interaction networks and biological pathways [
18]. Gene ontology of ETBR was obtained with a gene set enrichment tool and enrichment scores as described [
19].
Proof-of-concept: in vitro cytotoxic assays
To test whether endothelin receptor blockers affect GBM cell growth and toxicity in vitro, three drugs currently in use to treat pulmonary artery hypertension were used—ambrisentan (Letairis/Volibris), which selectively blocks the ETAR, and macitentan (Opsumit) and bosentan (Tracleer), which block both ETAR and ETBR—in standard viability assays with a CellTiter 96 Aqueous One Solution Cell Proliferation Assay system (Promega) as recommended by the manufacturer. We also tested BQ788, which is selective for ETBR, and ACT-132577, the active metabolite of macitentan. The ACT-132577, bosentan, BQ788 and macitentan used in this study were from Medchemexpress LCC (Princeton, NJ, USA), while ambrisentan was from Ark Pharm, Inc. (Libertyville, IL, USA). All the drugs were provided and checked by Medivir, Stockholm, Sweden. To test the effects of the drugs on cancer cells, we used primary GBM cells (GBM30, GBM42, GBM48, GBM392, and GBM398) and three GBM lines (U-251 MG, U-373 MG (Uppsala), and U-343 MGa). To test drug effects on normal cells, we used human umbilical vein endothelial cells (HUVECs, Lonza, CH-3930 Visp, Switzerland), MRC-5 lung fibroblasts (ATCC, LGC Standards, Middlesex, UK), and retinal pigment epithelial cells (a generous gift from Dr. Rich Stanton, Cardiff University). Since the endothelin axis (consisting of endothelins, ETAR, and ETBR) has also been implicated in breast cancer, we also tested the drugs on three breast cancer lines: MCF7, MDA-MA-231, and SK-BR-3. In brief, approximately 1 × 104 cells/well were seeded onto 96-well plates and treated with twofold serial dilutions of drug (0.78–200 μM). Cell viability was assessed on day 6 with a VersaMax ELISA Microplate Reader (Molecular Devices, Wokingham, Berkshire, UK) at an optical density of 490 nm (reference wave length, 650 nm). The optical density of treated cells was expressed as a percentage of untreated cells, which were considered 100% viable.
Statistical analysis
P < 0.05 was considered to indicate statistical significance. Survival curves were estimated with the Kaplan–Meier method, and the significance of differences between the curves was determined with the log-rank test. Boxplots were plotted with R programming and analyzed by t test.
Discussion
In this study, we investigated whether ETBR is overexpressed in GBM tumors in a Swedish patient cohort and assessed the potential usefulness of ETBR as a prognostic marker and drug target for GBMs and other types of cancer. We found that ETBR is indeed often overexpressed in GBM tumors, with little or no immunoreactivity in control brains. Analysis of expression data from TCGA and a subset of GEO datasets showed that overexpression of ETBR in GBM was correlated with shorter patient survival. Similarly, by examining ETBR expression across 470 cancers, glioma or GBM were again found to have high expression. By mapping the protein neighborhood to ETBR, we found that ETBR is mainly predicted to interact with eight proteins that further interact with 175 additional proteins, many of which are involved in cell-cell communication (gap junction, adherens junction), the vascular endothelial growth factor signaling pathway, and calcium signaling—all of which are associated with cancer pathogenesis. These results support the potential use of ETBR blockers as a targeted therapy for cancer [
10].
The endothelin axis has been implicated in the pathogenesis of many types of cancers (reviewed in [
23]). In particular, ETBR is overexpresssed in bladder carcinoma [
24], melanoma [
25], small-cell lung cancer [
26], vulvar cancer [
5], clear-cell renal cell carcinoma [
6], oesophageal squamous cell carcinoma [
7], and astrocytoma (including GBM) [
12]. ETBR was also earlier reported to be highly expressed in melanoma [
25]. Of note, ETBR overexpression was correlated with shorter patient survival or poor patient outcome in small-cell lung cancer, vulvar cancer, clear-cell renal cell carcinoma, esophageal squamous cell carcinoma, and GBM [
5‐
7,
12,
24,
27] and may thereby represent a potential prognostic marker as well as a therapeutic target for several cancer forms. We confirmed this hypothesis in the current study. We assessed the toxicity of ETBR and ETAR blockers for cancer cells of different origins. While Ambrisentan was not cytotoxic to GBM cells or breast cancer cells, the ETBR-selective blocker BQ788, the dual ETBR and ETAR blockers bosentan and macitentan, and the active metabolite of macitentan, ACT-132577 inhibited tumor cell growth to some extent. The affinity of ambrisentan to ETBR is at most 1% of its affinity to ETAR (IC
50 = 1 nM) (reviewed in [
28]), and ambrisentan was the least effective of the drugs we tested. We speculate that the ratio between ETAR and ETBR may be crucial for maximum cytotoxic efficiency.
To our knowledge, only two studies have earlier demonstrated overexpression of ETBR in GBM tumors, one study of Han-Chinese patients [
12] and one in Japanese patients [
29]. Ethnicity is a factor in the pathogenesis of gliomas [
13,
14]. Epidemiological data suggest that the incidence of glioma in the United States is higher among whites, followed by blacks, Hawaiians, Chinese or Japanese, Filipinos, and Alaskan natives [
30]. The incidence of gliomas is also higher in Scandinavian countries than in Asian countries [
30]. The ethnicity difference may be related to or result in alterations in the expression of key proteins. The promoter methylation status of the
O
6
-methylguanine methyltransferase, a key DNA repair enzyme predicted the outcome of treatments that include an alkylating agent in Caucasian populations but not in Indians [
31]. In the present study, we detected higher ETBR immunoreactivity in tumor cells from GBMs of Swedish patients, while little or no ETBR immunoreactivity was detected in adjacent nontumor cells, which consistent with a previous report [
20]. ETBR is known to predominantly express by astrocytes, where it helps regulate cell hypertrophy [
32]. In normal aging brains, we detected ETBR immunoreactivity in corpora amylacea, which are glycoproteinaceous inclusion bodies associated with aging or neurodegenerative diseases. The significance of this finding is unknown but it is well-known that the corpora amylacea is immunoreactive to various proteins (reviewed in [
33]) and recently, to an antibody against a late antigen (MAB8127, Millipore) of human cytomegalovirus, a ubiquitous beta herpesvirus [
34].
Our study is limited by the relatively small number of patients. Hence a large-scale patient cohort is needed to further evaluate the usefulness of ETBR as prognostic marker. A future study should also be tailored to better understand the role of ETBR in the pathogenesis of GBM. Nevertheless, our study confirms that ETBR is overexpressed in GBM and other cancer forms and further implicates ETBR as a potentially useful prognostic marker and possibly a therapeutic target for cancer.
Acknowledgements
We thank all lab members for valuable comments and Stephen Ordway for editorial assistance; Drs. Anna Martinez Casals and Mohsen Karimi Arzenani for the RNA and cDNA derived from GBM tissues, respectively; Anna Ridderstad Wollberg from VINNOVA-BIO-X for excellent coaching; Ylva Terelius, Richard Bethell, Fredrik Öberg and Susana Ayesa Alvarez from Medivir AB for useful technical advice and helped with the procurement and purity analysis of the endothelin antagonists.