|Astrocytoma is defined as the neoplasia of astrocytes, the most abundant non-neuronal glial cells in brain. According to the WHO classification, the different grades of astrocytoma are- gradeI/pilocytic astrocytoma (benign form), grade II/diffuse astrocytoma (DA), grade III/anaplastic astrocytoma (AA) and grade IV/Glioblastoma (GBM). Patients with grade II astrocytoma have median survival time of 6-8 years after surgical intervention. While the more aggressive grade III has a median survival of 2-3 years. Grade IV is the most malignant form and has a median survival of 15 months approximately. In spite of all the progress in the fields of diagnosis and therapy, the prognosis of GBM still remains very poor. The aggressiveness and poor survival of GBM is due to the recurrence which is primarily because of intratumoral heterogeneity, presence of glioma stem cells and infiltration of the tumor cells into the normal brain parenchyma. Apart from the role of genetic mechanisms in triggering tumorigenesis, epigenetic modifications particularly the DNA methylation and histone modifications, are now recognized as frequent alterations playing a crucial role in the development and progression of human malignancies. There are two distinct DNA methylation abnormalities. The first is the reduction in genome-wide DNA methylation levels (global hypomethylation) and the second is the hypermethylation in the CpG island of specific gene promoters. Hypomethylation is believed to induce proto-oncogene activation and chromosomal instability, whereas hypermethylation is strongly associated with silencing of tumor suppressor genes. Thus, DNA methylation can function as a “switch” to activate or repress gene transcription, providing an essential mechanism for overexpressed or silenced genes involved in the regulation of cell cycle, DNA repair, growth signalling, angiogenesis, apoptosis, migration, invasion and thus in the initiation and progression of astrocytoma.
Recent studies have identified biomarkers with prognostic impact which would include promoter methylation of O⁶-methyl guanine-DNA methyltransferase methylation (MGMT), isocitrate dehydrogenase 1(IDH1) mutation and a glioma CpG-island methylator (G-CIMP) phenotype. In the current study, we have characterized the DNA methylation profile for the different grades of astrocytoma and analysed the significance of methylation events occurring commonly in all the grades or uniquely only in grade IV. One of the GBM-specific hypomethylated and upregulated genes, Fibromodulin (FMOD), was extensively investigated in terms of its role in glioma pathogenesis and its regulation. FMOD was found to induce F-actin stress fibre formation and promote glioma cell migration. We also found that FMOD-mediated glioma cell migration is dependent on Integrin/FAK/Src/Small Rho GTPases signalling cascade. We further found that TGFβ pathway regulates FMOD expression through a process involving active demethylation and chromatin state transitions on FMOD promoter.
This work has been divided into three parts:
Part I: Characterization of DNA methylome during progression of Astrocytoma
To investigate the aberrant methylation pattern on a genome-wide scale, 17 Grade II, 16 Grade III and 36 Grade IV tumor samples as well as 9 control brain tissues were analysed using Infinium Human Methylation 450K Bead Array on Illumina platform. The analysis was carried out in two parts. Firstly, we validated the dataset with already existing TCGA dataset. Upon comparison, the methylation profile of our dataset was highly correlated to the TCGA dataset with correlation coefficient of 0.99. In addition, we also checked the methylation status of few known hypermethylated and hypomethylated genes which showed the similar type of differential methylation. Then, we characterized the differentially methylated CpGs based on their spatial distribution in the human genome, for different grades of astrocytoma. CpG-rich regions show more of hypermethylation while the non-CpG rich regions, like open sea or gene body, are observed to be hypomethylated. Secondly, we also analysed the differentially methylated genes which contribute to physiological events in gliomagenesis. We hypothesized that the methylation specific events that occur in grade II and remain similarly methylated in grade IV are the ones probably contributing to the initial astrocyte transformation. However, the methylation specific events responsible for the aggressive nature of grade IV may occur as differentially methylated genes only in grade IV (and not in grade II). In this analysis, we have identified differentially methylated genes that play a role in initial transformation process (293 genes hypermethylated and downregulated while 23 genes were hypomethylated and upregulated) and also those that play a role in tumor aggressiveness (459 genes hypermethylated and downregulated while 350 genes were hypomethylated and upregulated). The differentially methylated genes that were common in both grade II and grade IV showed an enrichment of cell proliferation pathways while the differentially methylated genes uniquely present in grade IV showed enrichment in pathways related to the aggressiveness phenotype of tumorigenesis like cell motility and angiogenesis.
Part II: Fibromodulin (FMOD), a GBM-specific hypomethylated and upregulated gene, is essential for glioma cell migration
Among differentially methylated genes specifically in GBM, fibromodulin (FMOD) is one of the top most hypomethylated genes. FMOD is a member of leucine – rich repeat proteoglycan that is widely distributed in interstitial connective tissues. We found that FMOD is hypomethylated and upregulated only in grade IV/GBM, not in the grade II. FMOD promoter methylation status is significantly negatively correlated to its transcript levels.Towards identifying functions of FMOD in glioma cells, total RNA derived from U251 cells transfected with either non-targeting siRNA or FMOD siRNA was subjected to transcriptome profiling. There were 872 genes upregulated and 299 genes downregulated in FMOD silenced cells than in control cells. PANTHER pathway analysis using the differentially regulated genes identified several pathways to be associated with FMOD. Cytoskeleton regulation by Rho GTPase, which is known to be involved in cell motility and migration, is enriched with highest significance. In coherence with the pathway analysis, modulating FMOD levels in glioma cells affected in glioma cell migration. Upon FMOD overexpression, there was significant increase in migration than in control cells. Conversely, when FMOD is silenced, there was delay in migration than in control cells and the delayed migration was rescued by the addition of recombinant purified FMOD protein. Prior neutralization with FMOD specific antibody inhibited cell migration suggesting that secreted FMOD promotes glioma cell migration. Overexpression of FMOD in glioma cells induced actin stress fibre formation required for the migration of cells. On the contrary, FMOD silencing resulted in the loss of F-actin stress fibres which was restored upon addition of FMOD purified protein exogenously to the media. To investigate further the role and the requirement of specific Rho GTPase in FMOD-mediated migration, each of members of Rho GTPase family was silenced and their effect on FMOD-induced silencing was studied. FMOD mediated glioma cell migration was delayed when RhoA, Rac1 and Cdc42 were silenced. In order to understand whether FMOD activates Integrin mediated signalling pathway, we performed western blot analysis to check the levels of phospho-FAK in either FMOD overexpressing or knockdown condition. We observed phospho-FAK levels increased upon FMOD overexpression and decreased upon FMOD silencing compared to the respective controls. Additional experiments revealed that inhibitors to Integrin, FAK and Src were able to abrogate the FMOD induced glioma cell migration. These results suggest that FMOD utilizes a pathway that involves Integrins, FAK, Src and Rho GTPases in promoting glioma cell migration. To comprehend the effect of FMOD promoter methylation status and its expression in GBM patient scenario, we stratified the patients into either high or low FMOD expression and promoter hypermethylation or hypomethylation. The GBM patients with low FMOD transcript levels and promoter hypermethylation showed better survival than the other group.
Part III: Regulation of FMOD expression through TGFβ-dependent epigenetic remodelling in glioma
To study how FMOD is regulated in glioma, we investigated the promoter sequence of FMOD by MatInspector. Several Smad-binding sites were located in FMOD promoter which indicated that FMOD might be regulated via TGFβ signalling pathway. Firstly, we checked active TGFβ signalling in glioma cell lines – LN229, U87 and U251. TGFβ-dependent signalling was active in U251 and U87 cells compared to LN229 cells as seen by the levels of phospho-Smad2. Moreover, FMOD transcript level was found to be high in U251 compared to LN229 cells. Further, TGFβ treatment increased FMOD promoter luciferase activity as well as FMOD transcript level in LN229 cells. In contrast, U251 cells that were treated with TGFβ RI inhibitor showed a significant decrease in FMOD promoter luciferase activity as well as FMOD transcript level. We correlated these findings with Smad2 occupancy at FMOD promoter by chromatin immunoprecipitation (ChIP). Smad2 association at FMOD promoter is found to be relatively higher in U251 cells than in LN229 cells which suggested that TGFβ induced transcription factor, Smad2, drives FMOD expression in U251 cells. Next, we investigated the role of TGFβ in FMOD promoter demethylation and chromatin state transition. Upon TGFβ treatment in LN229 cells, we found that there was gradual demethylation of FMOD promoter in a time-dependent manner. TGFβ treatment also altered the chromatin state by increasing the active marks (H3K4me3 and H3K9Ac) and decreasing the repressive mark (H3K27me3) with a simultaneous increase in Smad2 occupancy in the FMOD promoter. In contrast, TGFβ RI inhibitor treatment of U251 cells resulted in methylation of FMOD promoter in a time-dependent manner. Further, we observed a significant enrichment of repressive histone marks (H3K27me3) and loss of active chromatin marks (H3K4me3 and H3K9Ac) with a concomitant decrease in Smad2 occupancy at FMOD promoter. DNMT3A/B and EZH2 enzymes play a key role in DNA methylation and H3K27 trimethylation respectively. Accordingly, we examined the transcript levels of DNMT3A/B and EZH2 in LN229 cells treated with TGFβ as well as U251 cells treated with TGFβ RI inhibitor. In presence of TGFβ, DNMT3A/B and EZH2 transcript levels were significantly downregulated than in untreated cells in a time-dependent manner. Conversely, in U251 cells treated with TGFβ RI inhibitor, there was a significant increase in DNMT3A/B and EZH2 transcript levels when compared to untreated cells. TGFβ is known to promote glioma cell migration. In order to understand whether TGFβ-mediated glioma cell migration occurs via FMOD, we performed migration assay in U251 cells with or without TGFβ RI inhibitor followed by addition of either BSA control or FMOD purified protein. Upon TGFβ RI inhibitor treatment, there was delay in the migration of U251 cells than in untreated control cells which was rescued when purified FMOD protein was added, indicating that FMOD is essential for TGFβ signalling cascade to induce glioma cell migration. Therefore, we conclude from these results that epigenetically regulated FMOD is essential for TGFβ mediated glioma cell migration.