The glial cells of the brain and the peripheral nervous system retain the capacity to divide and proliferate throughout the lifespan of an individual and thereby have the propensity to give rise to the most adult neurological tumours. Among them, the tumours which arise from different kinds of glial cells are referred to as gliomas. Of the various types of gliomas, astrocytomas are the most common central nervous system neoplasms which make upto 60% of all the primary brain tumours. Being the most prevalent type, the WHO classifies them into grades ranging from I to IV based on their intensity of malignancy. Grade IV astrocytoma or Glioblastoma (GBM) is considered to be the most malignant form with a median survival of 14.6 months, in spite of all therapeutic modalities. GBM is further classified as primary and secondary GBM. Primary GBM manifests de novo without any early history of pre-malignant lesions, on the other hand secondary GBM arises progressively from lower grades over a period of 5-10 years. Like other malignancies, GBM also arises from various genetic and epigenetic variations. Epigenetic variations include all such mitotically and meiotically heritable traits that do not involve changes in DNA sequence. There are three major areas of epigenetics - DNA methylation, histone modifications and non-coding RNAs which are known to have profound effects on gene expression. A lot being known about the genetic derailments in GBM, in this study we looked into the epigenetic aspects of GBM. In our lab, we have carried out various high throughput studies, which unveiled the distorted landscape of DNA methylation and miRNA expression in GBM. This indicates that, in addition to the genetic mechanisms of gene alterations like mutations, copy number aberrations, protein coding genes are also affected by changes in methylation as well as by miRNA misregulation. The study has been divided into two parts. Part one of the study deals with the identification of chromobox homolog 7 (Cbx7), as a hypermethylated and downregulated gene in GBM. More importantly, Cbx7 is a member of the polycomb repressive complex and brings about its function through chromatin modifications. Here we have investigated the role of Cbx7 in gliomagenesis, and why it has to be silenced by methylation for tumorigenesis to ensue. In part two, we elucidated two unique ways of miRNA regulation in GBM. In the first section, we identified miR-326 as a PI3 kinase regulated miRNA and demonstrated its tumour suppressive role in GBM. In the other section, we analysed the copy number aberration data from TCGA and identified miR- 4484 as a miRNA subjected to deletion in GBM. We further went ahead to demonstrate its growth suppressive role in GBM. Part 1: Epigenetic regulation of the chromatin modifier Cbx7; chromobox homolog 7 DNA methylation is involved in the normal cellular control of expression and thereby plays a crucial role in maintaining the homeostasis of the cell. The phenomenon of DNA methylation keeps the various loci of the genome such as the germline specific genes and the repetitive transposable elements silenced, whereas the tumour suppressors and other growth modulator genes are spared from the methylation induced gene repression. One of the important steps that promote tumorigenesis is aberrant hypermethylation, which leads to the silencing of tumour-suppressor genes. Another important epigenetic phenomenon that affects the transcriptibility of the genome is histone modifications, which control the accessibility of the chromatin to the transcriptional machinery. In this section, we identified Cbx7, which happens to be an essential component of the chromatin modifying machinery, as an epigenetically regulated gene in GBM. We observed from the methylation array carried out in our lab, that Cbx7 was one of the highly methylated genes. We also validated that Cbx7 is downregulated in GBM and the same observation was further corroborated from other data sets. The hypermethylated state of Cbx7 was confirmed by DNA bisulphite sequencing and the expression levels of Cbx7 also got alleviated after 5-Aza-2′-deoxycytidine treatment, which is a DNA methylation inhibitor. This indicated that the down regulation of Cbx7 could be attributed to the methylation of its promoter region. In order to figure out the role of Cbx7 in GBM, we carried out transcriptome analysis of Cbx7 overexpressing cells compared to vector control condition by RNA sequencing. Gene ontology analysis revealed a significant enrichment of pathways involved in cell cycle, migration and invasion like processes. In fact, the exogenous overexpression of Cbx7 leads to cell death, reduced colony formation, retarded migration and invasion of cells. In order to explain the above phenotypes brought about by the exogenous expression of Cbx7, we further examined the RNA sequencing data and observed that many of the top most downregulated genes in Cbx7 overexpression state belonged to the Hippo signaling pathway. The effectors of the Hippo pathway, YAP and TAZ which essentially antagonize the pathway activity, are well known for their role in proliferation, migration and invasion in cancer. So we carried out a Gene Set Enrichment Analysis (GSEA) and found that there was a significant negative enrichment of YAP/TAZ targets in the Cbx7 regulated gene set. We validated some of these targets that were downregulated by Cbx7 overexpression. One of the most downregulated genes that we validated was Connective Tissue Growth Factor (CTGF), which also happens to be a bonafide target of YAP/TAZ. Independent downregulation of CTGF also resulted in reduced migration, thereby phenocopying the effects as were produced by Cbx7 overexpression. Moreover, we also observed that SAPK/JNK was the only kinase whose activity was abolished upon Cbx7 overexpression. Since CTGF is known to activate SAPK/JNK, we assessed the SAPK/JNK activity upon CTGF silencing. We found that levels of phospho-SAPK/JNK were significantly reduced in CTGF silenced condition. In addition to that, the inhibition of the SAPK/JNK by synthetic inhibitor also hampered the migration ability of the cells. We were also able to rescue the loss of migratory potential of glioma cells by the exogenous overexpression of CTGF in Cbx7 stable background. A similar rescue was also achieved by the overexpression of a constitutively active form of SAPK/JNK. This indicates that Cbx7 activates Hippo pathway to inhibit YAP/TAZ dependent transcription, resulting in the downregulation of CTGF, thereby inhibiting CTGF mediated activation of SAPK and thus resulting in the inhibition of glioma cell migration. PART 2: ROLE OF MIRNAS IN GLIOMA DEVELOPMENT AND PROGRESSION miRNAs are a class of small non-coding RNAs that are not translated into functional proteins but still contribute to numerous cellular processes, thereby adding yet another realm of regulation and control. miRNAs bring about gene regulation at the post-transcriptional level, either by degrading the mRNA or by translational repression and in this manner fine tune the expression of protein coding genes. miRNAs are often located in the most fragile sites of the genome which exposes them to grave genetic alterations, thus providing a circumstantial evidence of their etiological role in tumorigenesis. In a malignant state, miRNAs have been found to play pivotal roles in cellular transformation by altering various cellular phenotypes. Owing to their participation in diverse cellular functions, miRNAs have gained a strong foothold in gene regulation. Though a lot has been deciphered about the functional aspect of miRNAs, not much is known about the precise mechanisms which lead to their misregulation and therefore demands in-depth study. The expression of miRNAs can be modulated by a variety of genetic and epigenetic mechanisms. Section I: Role of miR-326 – a PI3 kinase regulated miRNA, in gliomagenesis The TCGA group in the year 2008 identified three major pathways which go disarray in GBM. These include the pro-tumorigenic receptor tyrosine kinase (RTK) pathway, and the p53 and the pRB tumour-suppressive pathways. The RTK signalling includes the PI3 kinase pathway, which is pivotal in gliomagenesis and many other cancers. This directed us to elucidate the set of miRNAs which are controlled by the aberrant functioning of the PI3 kinase pathway. We used synthetic inhibitor LY294002 to abrogate the PI3 kinase signalling and examined the miRNA profile in two glioma cell lines U87 and U251, which have an activated PI3 kinase pathway. Indeed the abrogation of the PI3 kinase pathway resulted in the modulation of a wide array of miRNAs. We validated miR-326 as one of the miRNAs that was upregulated upon PI3 kinase pathway abrogation. Furthermore, we observed that miR-326 was a down regulated miRNA in GBM and different glioma cell lines, as well as in many other publicly available data sets. We also observed that miR-326 is an intragenic miRNA and its host gene Arrestin β1 (ARRB1) also exhibited similar upregulation upon PI3K pathway inhibition. Over-expression of miR-326 resulted in various anti-tumorigenic affects like reduced proliferation, reduced migration and colony suppression. In order to find the targets of miR-326, we analysed the transcriptome by RNA sequencing upon pre-miR-326 transfection. We shortlisted and validated some of the genes which were getting regulated through miRNA over-expression and also explain the functional role of miR-326. Section II: Role of miR-4484 – a copy number deleted miRNA, in gliomagenesis In the TCGA study mentioned above, it was also unfurled that there are many genes in the RTK, p53 and pRB signalling pathways which are made dysfunctional through gene deletions and amplifications. We envisaged whether it is only the protein coding genes which are subjected to such regulations or the non-coding genes like miRNAs as well. In this pursuit, we identified miR-4484 as one of the miRNAs located in the deleted region of uroporphyrinogen III synthase (UROS) gene in the chromosome 11 of the GBM genome. As conceived, miR-4484 was observed to be a downregulated miRNA in association with its host gene UROS. We further elucidated that the downregulation was due to the co-deletion of a locus harbouring both the protein coding gene and the miRNA. In addition, upon over-expression of miR-4484, we observed reduced migration and colony formation, indicating its role as a tumour–suppressor. For seeking the targets of miR-4484, we extracted RNA from miR-4484 over-expression condition and subjected it to RNA sequencing. We shortlisted and validated some of the genes which were getting regulated through miRNA over-expression and possibly explain the functional role of miR-4484.

Glioma, the tumor of glial cells, is one of the common types of primary central nervous system (CNS) neoplasms. Astrocytoma is the most common of all gliomas and originates from astrocytic glial cells. Astrocytoma tumors belong to two main categories: benign tumors, comprising of grade I Pilocytic astrocytoma and malignant tumors which diffusely infiltrate throughout the brain parenchyma. Diffusely infiltrating astrocytomas are graded into diffuse astrocytoma (DA; grade II), anaplastic astrocytoma (AA; grade III) and glioblastoma (GBM; grade IV) in the order of increasing malignancy. Patients with grade II astrocytoma have a median survival time of 6 to 8 years after surgical intervention. While the more aggressive grade III (AA) and grade IV (GBM) are together called malignant astrocytomas, the treatment protocols and length of survival are distinctly different between these grades. The median survival time for grade III patients is 2 to 3 years whereas patients with grade IV have a median survival of 12-15 months. GBMs have been further divided into primary GBM and secondary GBM on the basis of clinical and histopathological criteria. Primary GBM presents in an acute de novo manner with no evidence of an antecedent lower grade tumor and it accounts for >90% of all GBMs. In contrast, secondary GBM results from the progressive malignant transformation of a grade II or grade III astrocytoma. The current WHO grading system of astrocytomas is based on the histopathological characteristics of the underlying tumor tissue. Diagnoses by pathologists are dependent on specific histologic features: increased mitosis, nuclear atypia, microvascular proliferation and/or necrosis, which associate with biologically aggressive behaviour (WHO 2007). Though grading based on histology is largely reproducible and well accepted, subjectivity involved and substantial disagreement between pathologists has remained a major concern. Because of inherent sampling problems (mainly due to tumor location in the brain) and inadequate sample size available for histological evaluation, there exists a very high possibility of error in grading. Recent studies have attempted to characterize the molecular basis for the histological and prognostic differences between grade III and grade IV astrocytoma. While reports have shown the grade specific profile of gene expression, there is no molecular signature that can accurately classify grade III and grade IV astrocytoma samples. In the current work, we have identified molecular signatures for the accurate classification of grade III and grade IV astrocytoma patients by using transcriptome and miRNome data. The receptor tyrosine kinase pathway is known to be overexpressed in 88% of glioblastoma patients. The expression and activation of the receptors is reported to be deregulated by events like amplification and activating mutations. The aberrant expression of RTKs could also be due to the deregulation of miRNAs, which, in the untransformed astrocytes regulate and fine-tune the levels of the RTKs. In the current study, we have identified that tumor suppressor miRNA miR-219-5p regulates RTK pathway by targeting EGFR and PDGFRα. Part I. Transcriptome approach: Identification of a 16-gene signature for classification of malignant astrocytomas In order to obtain a more robust molecular classifier to accurately classify grade III and grade IV astrocytoma samples, we used transcriptome data from microarray study previously performed in our laboratory. The differential regulation of 175 genes identified from microarray was validated in a cohort of grade III and grade IV patients by real-time qRT-PCR. In order to identify the classification signature that can classify grade III and grade IV astrocytoma samples, we used the expression data of 175 genes for performing Prediction Analysis of Microarrays (PAM) in the training set of grade III and grade IV astrocytoma samples. PAM analysis identified the most discriminatory 16-gene expression signature for the classification of grade III and grade IV astrocytoma. The Principal Component Analysis (PCA) of 16-genes astrocytoma patient samples revealed that the expression of 16-genes could classify grade III and grade IV astrocytoma samples into two separate clusters. In the training set, the 16-gene signature was able to classify grade III and grade IV patients with an accuracy rate of 87.9% as tested by additional analysis of Cross-Validated probability by PAM. The 16-gene signature obtained in the training set was validated in the test set with diagnostic accuracy of 89%. We further validated the 16-gene signature in three independent cohorts of patient samples from publicly available databases: GSE1993, GSE4422 and TCGA datasets and the classification signature got validated with accuracy rates of 88%, 92% and 99% respectively. To address the discordance in grading between 16-gene signature and histopathology, we looked at the clinical features (age and survival) and molecular markers (CDKN2A loss, EGFR amplification and p53 mutation) that differ substantially between grade III and grade IV in discordant grade III and grade IV samples. The grading done by 16-gene signature correlated with known clinical and molecular markers that distinguish grade III and grade IV proving the utility of the 16-gene signature in the molecular classification of grade III and grade IV. In order to identify the pathways that 16 genes of the classification signature could regulate, we performed protein-protein interaction network and subsequently pathway analysis. The pathways with highest significance were ECM (extracellular matrix) and focal adhesion pathways, which are known to be involved in the epithelial to mesenchymal transition (EMT), correlating well with the aggressive infiltration of grade IV tumors. In addition to accurately classifying the grade III and grade IV samples, the 16-gene signature also demonstrated that genes involved in epithelial-mesenchymal transition play key role in distinguishing grade III and grade IV astrocytoma samples. Part II. miRNome approach microRNAs (miRNAs) have emerged as one of the important regulators of the interaction network that controls various cellular processes. miRNAs are short non-coding RNAs (mature RNA being 21-22nt long) that regulate the target mRNA by binding mostly in the 3’ UTR bringing about either translational repression or degradation of the target. miRNAs are shown to play key roles in cell survival, proliferation, apoptosis, migration, invasion and various other characteristic features that get altered in human cancers. miRNAs are characterized to have oncogenic or tumor suppressor role and the aberrant expression of miRNAs is reported in multiple human cancer types. Part A. Genome-wide expression profiling identifies deregulated miRNAs in malignant astrocytoma With an aim to identify the role of miRNAs in the development of in malignant astrocytoma, we performed a large-scale, genome-wide microRNA (miRNA) (n=756) expression profiling of 26 grade IV astrocytoma, 13 grade III astrocytoma and 7 normal brain samples. Using Significance Analysis of Microarrays (SAM), we identified several differentially regulated miRNAs between control normal brain and malignant astrocytoma, grade III and grade IV astrocytoma, grade III astrocytoma and grade IV secondary GBM, progressive pathway and de novo pathway of GBM development and also between primary and secondary GBM. Importantly, we identified a most discriminatory 23-miRNA expression signature, by using PAM, which precisely distinguished grade III from grade IV astrocytoma samples with an accuracy of 90%. We re-evaluated the grading of discordant samples by histopathology and identified that one of the discordant grade III samples had areas of necrosis and it was reclassified as grade IV GBM. Similarly, out of two discordant grade IV samples, one sample had oligo component and it was reclassified as grade III mixed oligoastrocytoma. Thus, after the revised grading, the prediction accuracy increased from 90% to 95%. The differential expression pattern of nine miRNAs was further validated by real-time RT-PCR in an independent set of malignant astrocytomas (n=72) and normal samples (n=7). Inhibition of two glioblastoma-upregulatedmiRNAs (miR-21 and miR-23a) and exogenous overexpression of two glioblastoma-downregulatedmiRNAs (miR-218 and miR-219-5p) resulted in reduced soft agar colony formation but showed varying effects on cell proliferation and chemosensitivity. Thus, we have identified the grade specific expression of miRNAs in malignant astrocytoma and identified a miRNA expression signature to classify grade III astrocytoma from grade IV glioblastoma. In addition, we have demonstrated the functional relevance of miRNA modulation and thus showed the miRNA involvement and their importance in astrocytoma development. Part B. miR-219-5p inhibits the receptor tyrosine kinase pathway by targeting mitogenic receptor kinases in glioblastoma The receptor tyrosine kinase (RTK) pathway, being one of the important growth promoting pathways, is known to be deregulated in 88% of the patients with glioblastoma. In order to understand the role of miRNAs in regulating the RTK pathway, we undertook a screening procedure to identify the potential miRNAs that could target different members of the RTK pathway. From the screening study involving bioinformatical prediction of miRNAs and subsequent experimental validation by modulation of miRNA levels in glioma cell lines, we identified miR-219-5p as a candidate miRNA. The overexpression of miR-219-5p reduced the protein levels of both EGFR and PDGFRα. We confirmed the binding of miR-219-5p to the 3’ UTRs by using reporter plasmids. We also confirmed the specificity of miR-219-5p binding sites in the 3’ UTR of EGFR by site directed mutagenesis of binding sites which abrogated the miRNA-UTR interaction. The expression of miR-219-5p was significantly downregulated in grade III as well as in grade IV astrocytoma samples in the miRNA microarray experiment and we further validated the downregulation in an independent cohort of grade III and grade IV astrocytoma patients by real-time qRT-PCR. The ectopic overexpression of miR-219-5p in glioma cell lines inhibited cell proliferation, colony formation, anchorage independent growth and the migration of glioma cells. In addition, overexpression of miR-219-5p decreased MAPK and PI3K pathways, in concordance with its ability to target EGFR and PDGFRα. Additionally, for the further characterization of miR-219-5p – EGFR interaction and its effect on MAPK and PI3K pathways, we used U87 glioma cells that stably overexpress wild-type EGFR and constitutively active ΔEGFR (both lacking 3’-UTR and thus being insensitive to miR-219-5p overexpression) along with U87 parental cells. In these cell lines with the overexpression of EGFR lacking 3’-UTR, miR-219-5p was unable to inhibit - MAPK and PI3K pathways and also glioma cell migration suggesting that these effects were indeed because of its ability to target EGFR. Further, in the glioblastoma patient cohort (TCGA dataset), we found significant negative correlation between EGFR protein levels, both total EGFR and phospho EGFR and miR-219-5p levels in the glioblastoma tissue samples suggesting a role of miR-219-5p in increasing the protein levels of EGFR in glioblastoma. In summary, we have identified and characterized miR-219-5p as the RTK regulating tumor suppressor miRNA in glioblastoma.