Cell lines derived from tumor tissues have been used as a valuable system to study gene regulation and cancer development. Comprehensive characterization of the genetic background of cell lines could provide clues on novel genes responsible for carcinogenesis and help in choosing cell lines for particular studies. Here, we have carried out whole exome and RNA sequencing of commonly used glioblastoma (GBM) cell lines (U87, T98G, LN229, U343, U373 and LN18) to unearth single nucleotide variations (SNVs), indels, differential gene expression, gene fusions and RNA editing events. We obtained an average of 41,071 SNVs out of which 1,594 (3.88%) were potentially cancer-specific. The cell lines showed frequent SNVs and indels in some of the genes that are known to be altered in GBM- EGFR, TP53, PTEN, SPTA1 and NF1. Chromatin modifying genes- ATRX, MLL3, MLL4, SETD2 and SRCAP also showed alterations. While no cell line carried IDH1 mutations, five cell lines showed hTERT promoter activating mutations with a concomitant increase in hTERT transcript levels. Five significant gene fusions were found of which NUP93-CYB5B was validated. An average of 18,949 RNA editing events was also obtained. Thus we have generated a comprehensive catalogue of genetic alterations for six GBM cell lines.

The most common and aggressive form of primary brain tumor in adults is glioblastoma (GBM). From the global DNA methylation profiling study, previously published from our laboratory, we identified Guanine Nucleotide binding-protein Gamma subunit 4 (GNG4) to be one of the most hyper methylated and down regulated genes in GBM. GBM derived cell lines showed reduced GNG4 transcript levels, which could be reversed by methylation inhibitor treatment. Bisulphite sequencing confirmed the methylation status in glioblastoma tumor tissue and GBM derived cell lines. Overexpression of GNG4 was found to inhibit proliferation and colony formation of GBM cell lines and in vitro transformation of immortalized human astrocytes, thus suggesting a potential tumor suppressor role of GNG4 in GBM. Correlation of GNG4 transcript levels with that of all GPCRs from TCGA data revealed chemokine receptors as the potential target of GNG4. Furthermore, exogenous over expression of GNG4 inhibited SDF1α/CXCR4-dependent chemokine signaling as seen by reduced pERK and pJNK and GBM cell migration. The inhibitory association between GNG4 and SDF1α/CXCR4 was more evident in mesenchymal subtype of GBM. Thus, this study identifies GNG4 as an inhibitor of SDF1α/CXCR4-dependent signaling and emphasizes the significance of epigenetic inactivation of GNG4 in glioblastoma, especially in mesenchymal subtype.

Despite significant advances in the understanding of the biology, the prognosis of glioblastoma (GBM) remains dismal. The objective was to carry out whole-exome sequencing (WES) of Indian glioma and integrate with that of TCGA to find clinically relevant mutated pathways. WES of different astrocytoma samples ( = 42; Indian cohort) was carried out and compared with that of TCGA cohort. An integrated analysis of mutated genes from Indian and TCGA cohorts was carried out to identify survival association of pathways with genetic alterations. Patient-derived glioma stem-like cells, glioma cell lines, and mouse xenograft models were used for functional characterization of calcitonin receptor (CALCR) and establish it as a therapeutic target. A similar mutation spectrum between the Indian cohort and TCGA cohort was demonstrated. An integrated analysis identified GBMs with defective "neuroactive ligand-receptor interaction" pathway ( = 23; 9.54%) that have significantly poor prognosis ( < 0.0001). Furthermore, GBMs with mutated calcitonin receptor () or reduced transcript levels predicted poor prognosis. Exogenously added calcitonin (CT) inhibited various properties of glioma cells and pro-oncogenic signaling pathways in a CALCR-dependent manner. Patient-derived mutations in CALCR abolished these functions with the degree of loss of function negatively correlating with patient survival. WT CALCR, but not the mutant versions, inhibited Ras-mediated transformation of immortalized astrocytes Furthermore, calcitonin inhibited patient-derived neurosphere growth and glioma tumor growth in a mouse model. We demonstrate CT-CALCR signaling axis is an important tumor suppressor pathway in glioma and establish CALCR as a novel therapeutic target for GBM. .

Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation-specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation.

Background Gliomas are the most common and lethal type of intracranial tumors. With the current treatment regime, the median survival of patients with grade IV glioma (glioblastoma/GBM) remains at 14–16 months. RNA editing modifies the function and regulation of transcripts. The development of glial tumors may be caused by altered RNA editing events. Methods In this study, we uncover the global RNA editome landscape of glioma patients from RNA-seq data of control, lower grade glioma (LGG) and GBM samples (n = 1,083). Results A-to-I editing events were found to comprise 80% of the total editing events of which 96% were located in the Alu regions. The total RNA editing events were found to be reduced in glioma compared to control samples. More specifically, we found Gamma-aminobutyric acid type A receptor alpha3 (GABRA3) to be edited (c.1026 A-to-G; pI343M) in 73% (editing ratio 0.8) of control samples compared to LGG (28.96%; 0.47) and GBM (5.2%; 0.53) samples. GABRA3 transcript level was found to be downregulated in glioma compared to control in a grade-specific manner with GBMs having the lowest level of the transcript. Further, GABRA3 transcripts were observed to be higher in edited compared to unedited glioma samples. The transcript and protein levels of exogenously expressed gene were found to be higher for edited compared to unedited GABRA3 in glioma cells. Further, exogenously expressed edited GABRA3 inhibited migration and invasion of glioma cells efficiently but not the unedited GABRA3. Conclusion Collectively, our study discovered a reduction in RNA editing during glioma development. We further demonstrate that elevated RNA editing maintains a high level of GABRA3 RNA and protein in normal glial cells which provides a less migratory environment for the normal functioning of the brain. In contrast, the reduction in GABRA3 protein levels, due to lower stability of unedited RNA, results in the loss of function which confers an aggressive phenotype to GBM tumor.