BMP4, a strong better prognosis predictor, has a subtype preference and cell development association in gliomas
- Zhaoshi Bao†1,
- Chuanbao Zhang†1,
- Wei Yan1,
- Yanwei Liu1,
- Mingyang Li1,
- Wei Zhang1Email author and
- Tao Jiang1, 2Email author
© Bao et al.; licensee BioMed Central Ltd. 2013
Received: 28 February 2013
Accepted: 11 April 2013
Published: 16 April 2013
The bone morphogenetic family proteins (BMP) are phytogenetically conserved proteins, which are essential for embryonic development. The key regulatory subunit, the bone morphogenetic protein 4 (BMP4), is overexpressed and associated with tumor metastasis in a variety of cancers. However, the prognostic and molecular features of gliomas with BMP4 expression is still unclear.
We obtained whole genome mRNA expression microarray data of 220 glioma samples of all grades from Chinese Glioma Genome Atlas (CGGA) database (http://www.cgga.org.cn) as discovery set. Of the 123 high-grade gliomas in this set, 33 Grade III tumors and 88 GBMs were analyzed by Kaplan-Meier method. Immunohistochemistry was used for validating the expression of BMP4 in another 77 glioma samples. Three additional datasets were obtained as validation sets. Gene ontology (GO) analysis and gene set variation analysis (GSVA) were used for functional annotation of BMP4.
In the discovery set, BMP4 overexpression was significantly associated with low grade as well as the lower mortality of high-grade gliomas in survival analysis (log-rank, p<0.05 in GBM patients and p<0.01 in anaplastic gliomas, respectively). BMP4 also showed a Proneural subtype, G1 subtype and Isocitrate Dehydrogenase 1 (IDH1) mutation preference and cell development association. The results of validation 4 datasets showed similar findings. The overexpression of BMP4 was also detected in low grade gliomas compared to the high grade ones by immunohistochemistry (p<0.05, chi-square test).
BMP4 expression was independently associated with grade and good prognosis in grade III and grade IV gliomas, suggesting BMP4 as a novel biomarker with potential important therapeutic implications.
Glioma is the most common type of brain tumor and is an important cause of cancer related mortality among adults and children . It can be divided into low grade glioma (LGG) and high grade glioma (HGG) depending on the malignancy. The median survival of patients with primary glioblastoma (GBM), the most malignant and frequent type of glioma, is approximately 1 year. But because of the heterogeneity of cancer, it varies remarkably from <1 week to >3 years after diagnosis , suggesting the limitations of the current diagnostic, predictive and prognostic markers and better therapeutic strategies are in the urgent need.
The introduction of molecular biomarkers in the management of patients with cancer may improve their clinical outcomes. The bone morphogenetic family proteins (BMP) are phytogenetically conserved proteins, which are essential for embryonic development [3, 4]. Their key regulatory subunit, the bone morphogenetic protein 4 (BMP4), is overexpressed and associated with pathogenesis and metastasis in a variety of cancers [5–7]. However, the prognostic and molecular features of gliomas with BMP4 expression is still unclear.
In this study, We obtained whole genome mRNA expression microarray data of 220 glioma samples of all grades from Chinese Glioma Genome Atlas (CGGA) database (http://www.cgga.org.cn) as discovery set  and 3 additional previously published datasets as validation sets. After studying the expression level of BMP4 in these samples, we analyzed the prognostic value of it. The expression difference was validated in another 77 glioma samples from Chinese Glioma Tissue Database (CGTD) by Immunohistochemistry. We also performed function annotation of BMP4 by GO analysis and GSVA, which revealed its correlation with cell development, differentiation and biogenesis.
Datasets used in this study
Whole genome mRNA expression microarray data and clinical information of 220 glioma samples of all grades from Chinese Glioma Genome Atlas (CGGA) database  (http://www.cgga.org.cn) were obtained as discovery set and 202 glioma expression files from the cancer genome atlas (TCGA) database  (http://cancergenome.nih.gov), the Repository for Molecular Brain Neoplasia Data (REMBRANDT, https://caintegrator.nci.nih.gov/rembrandt/) and GSE16011 data  (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE16011) were obtained as validation sets.
Gene ontology (GO) analysis of BMP4 associated genes
After Pearson correlation analysis, gene ontology analysis of the positively correlated genes (r>0.4, p<0.05) were analyzed by DAVID (http://david.abcc.ncifcrf.gov/home.jsp).
GSVA with BMP4 expression
Briefly, Immunoperoxidase staining for BMP4 (Abcam, ab39973) were performed following the standard protocol recommended by the manufacturer. Each slide stained for and BMP4 was individually reviewed and scored by two independent observers. Discrepancies in scoring between the two observers were resolved by additional review of the specimens and discussion between the reviewers until a consensus was achieved. Approximately 15-20 fields at 400× magnification were analyzed per specimen. The proportion of positively stained tumor cells was graded as follows: 0, no positive tumor cells; 1, <5% positive tumor cells; 2, 5-20% positive tumor cells; and 3, >20% positive tumor cells. The intensity of staining was recorded on a scale of 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown) and 3 (strong staining, brown). The staining index was calculated as follows: staining index = staining intensity × tumor cell staining grade. High BMP4 expression was defined as a staining index score ≥4, while low expression was defined as a staining index <4.
For molecular subtype annotation of the 4 datasets, we applied prediction analysis of microarrays (PAM) as previously reported . Quantitative results were shown as mean ± standard deviation. The difference of BMP expression was tested by the Student t-test in microarray data and by chi-square test in IHC results. Overall survival time (OS) was calculated from the date of diagnosis until death or the last follow-up. The survival curve of patients with high or low expressed BMP4 was calculated with the Kaplan-Meier method and the difference was analyzed using the two-sided log-rank test. A p-value < 0.05 was considered statistically significant. All the data analysis was performed in GraphPad Prism and R.
Written informed consent was obtained from the patient for publication of this report and any accompanying images. The study was performed with the approval of Ethics Committee of Capital Medical University and was in compliance with the Helsinki Declaration.
Characteristics of patients included
Clinical characteristics of 220 glioma patients
Median OS (days)
ND, not determined
BMP4 was overexpressed in low-grade gliomas
The expression level of BMP4 was validated in an independent group of patients by IHC
BMP4 was a better prognostic marker in anaplastic gliomas and glioblastomas
BMP4 expression showed a subtype preference
BMP4 was associated with cell development
Top 20 GO terms of BMP4 positively associated genes
BMP signaling pathway
regulation of astrocyte differentiation
regulation of glial cell differentiation
regulation of gliogenesis
regulation of cell development
negative regulation of astrocyte differentiation
transmembrane receptor protein serine/threonine kinase signaling pathway
negative regulation of cell development
regulation of striated muscle tissue development
regulation of muscle development
regulation of neurogenesis
regulation of transcription
negative regulation of glial cell differentiation
negative regulation of gliogenesis
regulation of nervous system development
middle ear morphogenesis
pattern specification process
The positive correlation of astrocytic genes and BMP4
Glioma is the most common lethal intracranial tumor in adults. Even after years of efforts in developing and improving therapeutic strategies, the glioma patients still have a poor survival time. As the identification of novel biomarkers and new molecular classification systems, we really see the dawn of a new era.
The bone morphogenetic family proteins (BMP) are highly conserved proteins, which are essential for embryonic development. Since potent developmental regulators are frequently disrupted in cancer , it is to be expected that BMP4 also contributes to tumor development. In recent years, important new advances has been generated on the contribution of BMP family members, such as BMP4, in cancer pathogenesis. Firstly, BMP4 gene variants have been shown to predispose to colorectal cancer . Meantime, the expression level of BMP4 are frequently altered in many tumor types [5–7, 16, 17]. Both in vivo and in vitro studies have demonstrated the role of BMP4 on suppression of cell growth [18, 19], induction to migration, invasion and epithelial-mesenchymal transition [20, 21], which are associated with cancer metastasis and progression.
There are only limited reports on the role of BMP4 in gliomas focusing on the anti-proliferation effect of BMP4 to stem-like cells [22–25] and GBM cell lines .In the present study, we found that BMP4 was overexpressed in LGGs. The expression difference was validated by microarray data from other datasets and IHC results from an independent group of patients from CGTD. These results showed that BMP4 was a potential marker for grading of gliomas.
Considering its effects on growth suppression, BMP4 has been suggested as a possible therapeutic target in cancer cells. Nevertheless, the other functional characteristics of BMP4, especially the promotion of cell mobility, make such strategies less appealing. Improved knowledge of the downstream mediators of BMP4 effects in cancer cells may allow dissection of the different BMP4 induced phenotypes and thereby generation of specific targeted therapies .
For prognosis analysis, we generated survival curve of data from CGGA and 3 other datasets by Kaplan-Meier method and the difference was analyzed using the two-sided log-rank test. Patients with higher expression of BMP4 showed a significantly better prognosis in anaplastic gliomas and GBMs. That exhibited the predomination of beneficial effects over detrimental effects of BMP4 in gliomas. Hence, BMP4 showed its capacity to be a better prognostic marker and a therapeutic target. We also found the preference expression of BMP4 in IDH1 mutation patients, Proneural subtype or G1 subtype, which was in concordance with the better prognosis of BMP4 overexpressed patients.
BMP4 was preferentially expressed in LGGs, IDH1 mutation patients, Proneural subtype and G1 subtype. And it was associated with the better prognosis in grade III and grade IV gliomas, all of which suggested that BMP4 was a novel biomarker with potential important therapeutic implications.
This work was supported by grants from 1. National High Technology Research and Development Program (No.2012AA02A508) 2. International Science and Technology Cooperation Program (No. 2012DFA30470) 3. National Natural Science Foundation of China (No. 81201993) 4. National 973 program (No.2011CB707804).
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