Добірка наукової літератури з теми "Glioma Stem-like Cells"

Abstract Gliomas are the most frequently diagnosed human primary brain tumors. Mutations in Isocitrate Dehydrogenase (IDH) 1 occur in the vast majority of low grade gliomas and secondary high grade glioblastomas. A single amino acid missense mutation in IDH1 at arginine 132 (R132H) is an early event in tumor development. IDH1R132H leads to the production of the oncometabolite 2-R-2-hydroxyglutarate. However the exact roles played by IDH1R132H in the development and malignant transformation of the tumors remain unclear. Further studies are required to determine optimal therapeutic strategies to target the IDH1 mutated subsets of gliomas. New generation high-throughput genetic perturbation technologies make it possible to systematically identify the genes and pathways required for the survival and proliferation of mammalian cells. Herein, we present preliminary results from a CRISPR-dCas9 derived activation to drive the transcriptional activation of more than 23,000 coding genes in both wild type and mutant IDH1 patient-derived pediatric glioma cells. Based on an average of three viability screens per cell type, we analyzed the sgRNA library representation in the IDH1 mutated and non-mutated glioma cultures after the genome wide activation. We identified 1553 candidate genes that upon gain of function trigger the death of glioma cells harboring the IDH1 mutation. The analysis of these results further pinpoints the activation of the Cyclin E1 (CCNE1), the BCL2 Antagonist/Killer 1 (BAK1) and the Homeobox B13 (HOXB13) as the most significant synthetic lethal targets in IDH1R132H glioma cells. Interestingly, results from RNAseq showed a decreased expression of these genes in IDH1 mutated compared to non-mutated glioma cells. Thus, this viability screening aims to elucidate genes that interact with IDH1R123H and play a role in tumor cell fitness. The functional analysis of these candidate genes will allow us to uncover their contribution to the progression of IDH1 mutated gliomas.

Diffuse gliomas continue to be an important problem in neuro-oncology. To solve it, studies have considered the issues of molecular pathogenesis from the intratumoral heterogeneity point. Here, we carried out a comparative dynamic analysis of the different cell populations’ content in diffuse gliomas of different molecular profiles and grades, considering the cell populations’ functional properties and the relationship with patient survival, using flow cytometry, immunofluorescence, multiparametric fluorescent in situ hybridization, polymerase chain reaction, and cultural methods. It was shown that an increase in the IDH-mutant astrocytomas and oligodendrogliomas malignancy is accompanied by an increase in stem cells’ proportion and mesenchymal cell populations’ appearance arising from oligodendrocyte-progenitor-like cells with cell plasticity and cells’ hypoxia response programs’ activation. In glioblastomas, malignancy increase is accompanied by an increase in both stem and definitive cells with mesenchymal differentiation, while proneuronal glioma stem cells are the most likely the source of mesenchymal glioma stem cells, which, in hypoxic conditions, further give rise to mesenchymal-like cells. Clinical confirmation was a mesenchymal-like cell and mesenchymal glioma stem cell number, and the hypoxic and plastic molecular programs’ activation degree had a significant effect on relapse-free and overall survival. In general, we built a multi-vector model of diffuse gliomas’ pathogenetic tracing up to the practical plane.

Object Recurrent malignant gliomas have inherent resistance to traditional chemotherapy. Novel therapies target specific molecular mechanisms involved in abnormal signaling and resistance to apoptosis. The proteasome is a key regulator of multiple cellular functions, and its inhibition in malignant astrocytic lines causes cell growth arrest and apoptotic cell death. The proteasome inhibitor bortezomib was reported to have very good in vitro activity against malignant glioma cell lines, with modest activity in animal models as well as in clinical trials as a single agent. In this paper, the authors describe the multiple effects of bortezomib in both in vitro and in vivo glioma models and offer a novel explanation for its seeming lack of activity. Methods Glioma stem-like cells (GSCs) were obtained from resected glioblastomas (GBMs) at surgery and expanded in culture. Stable glioma cell lines (U21 and D54) as well as temozolomide (TMZ)-resistant glioma cells derived from U251 and D54-MG were also cultured. GSCs from 2 different tumors, as well as D54 and U251 cells, were treated with bortezomib, and the effect of the drug was measured using an XTT cell viability assay. The activity of bortezomib was then determined in D54-MG and/or U251 cells using apoptosis analysis as well as caspase-3 activity and proteasome activity measurements. Human glioma xenograft models were created in nude mice by subcutaneous injection. Bevacizumab was administered via intraperitoneal injection at a dose of 5 mg/kg daily. Bortezomib was administered by intraperitoneal injection 1 hour after bevacizumab administration in doses of at a dose of 0.35 mg/kg on days 1, 4, 8, and 11 every 21 days. Tumors were measured twice weekly. Results Bortezomib induced caspase-3 activation and apoptotic cell death in stable glioma cell lines and in glioma stem-like cells (GSCs) derived from malignant tumor specimens Furthermore, TMZ-resistant glioma cell lines retained susceptibility to the proteasome inhibition. The bortezomib activity was directly proportional with the cells' baseline proteasome activity. The proteasome inhibition stimulated both hypoxia-inducible factor (HIF)–1α and vascular endothelial growth factor (VEGF) production in malignant GSCs. As such, the VEGF produced by GSCs stimulated endothelial cell growth, an effect that could be prevented by the addition of bevacizumab (VEGF antibody) to the media. Similarly, administration of bortezomib and bevacizumab to athymic mice carrying subcutaneous malignant glioma xenografts resulted in greater tumor inhibition and greater improvement in survival than administration of either drug alone. These data indicate that simultaneous proteasome inhibition and VEGF blockade offer increased benefit as a strategy for malignant glioma therapy. Conclusions The results of this study indicate that combination therapies based on bortezomib and bevacizumab might offer an increased benefit when the two agents are used in combination. These drugs have a complementary mechanism of action and therefore can be used together to treat TMZ-resistant malignant gliomas.

Abstract Primary gliomas arising within the brain remain the deadliest form of brain cancer and account for 78% of all malignant brain tumors. Glioblastoma patients have a 5 percent five-year survival rate and drug-resistant tumors often recur following surgical resection and treatment with radiation or chemotherapy. The cancer stem cell hypothesis suggests the presence of a subset of undifferentiated cells, namely glioma stem cells (GSCs), in the heterogenous tumor mass that are likely responsible for tumor initiation and recurrence of tumors post resection and give rise to drug resistant tumors. Determining a way to suppress these malignant characteristics and/or depleting the GSC population could improve current cancer treatments and the survival of glioma patients. Some GSCs are similar to oligodendrocyte precursor cells (OPCs), found during neural development and also residing in the adult brain. OPCs are prone to malignant transformation and believed to be a cellular origin for glioma. Recent findings indicate that the well-characterized neurotransmitter acetylcholine (ACh) maintains the primitive state of normal OPCs via muscarinic ACh receptors (mAChRs) preventing maturation and cell cycle exit. We investigated the functional characterization of ACh and mAChRs in modulating malignancy in OPC-like cells. We studied cultures of primary mouse OPC-like GSCs. We used the primary cells to culture three-dimensional tumor organoids in vitro to better represent tumor heterogeneity. We also used patient derived xenografts (PDX) of glioma to grow flank xenografts in NSG mice for an in vivo model. Publicly available data and studies in our lab show high levels of expression of CHRM3 (M3mAChR) in glioma patients and in primary OPC like GSCs. A drug screen conducted in the context of multiple sclerosis determined that benztropine (BZT), an anti muscarinic drug targeting M3mAChR, causes normal OPCs to exit the cell cycle, lose stem cell like characteristics, and differentiate. In our lab, electrophysiological studies demonstrated that activation of mAChRs in OPC-like cells from mouse and patient tumors generates rapid (< 1 second) increases in cytosolic calcium. Pharmacologic studies indicated that treatment with FDA approved anti muscarinic benztropine suppresses proliferation in cultured glioma cells. We also observed that serially passaging flank tumors treated with BZT into new host mice slowed down recurrence of new tumors. In order to determine key receptors mediating cholinergic responses in OPC-like cells we performed protein quantification, which displayed lowered phosphorylation of ERK. Electrophysiological studies are being conducted to dissect the mechanisms by which ACh evokes calcium release. These studies improve understanding of how cholinergic microenvironment influences stem-like glioma cells, providing a platform for repositioning available small molecule modifiers for treatment of glioma. Citation Format: Sumyuktha V. Anand, Alexander G. Skorput, Allan T. Gulledge, Isabella B. Fox, Damian A. Bonnin, Alison L. Young, Matthew C. Havrda. Targeting muscarinic acetylcholine receptors in glioma stem like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 905.

Abstract Gliomas are invasive cancers that resist all forms of attempted therapy, but immunotherapy has improved survival for some patients. We present evidence that another level of complexity may also contribute to lack of responses by the lymphocytes towards gliomas. Atomic force microscopy of four different glioma types: human U251 and rat T9 and F98 glioma cells, including freshly isolated human GBM neurosphere cultures (containing “stem cell-like cells’), revealed a complex surface topography with numerous microvilli and filopodia. These structures were not found on other cell types. Electron microscopy and immunofluorescence microscopy of glioma cells confirmed that microvilli are present. U251 cells with microvilli resisted the cytolytic actions of different human effector cells, (lymphokine-activated killer cells, γδ T cells, conventional cytolytic T lymphocytes, [CTL], and chimeric antigen receptor redirected T cells) better than their non-microvilli expressing counterparts. Killer cells released perforin which was detected within the glioma’s microvilli/ filopodia, indicating these structures can receive the cytolytic effector molecules, but cytotoxicity is suboptimal. Transfection of fascin siRNA into U251 cells prevented the microvilli from forming and allowed cytolytic cells to kill these adherent cells just as well as the non-attached glioma cells. These microvilli play multiple roles in glioma biology, such as invasion and resistance to lymphocyte-mediated killing.

Glioblastoma (GBM) still presents as one of the most aggressive tumours in the brain, which despite enormous research efforts, remains incurable today. As many theories evolve around the persistent recurrence of this malignancy, the assumption of a small population of cells with a stem-like phenotype remains a key driver of its infiltrative nature. In this article, we research Chordin-like 1 (CHRDL1), a secreted protein, as a potential key regulator of the glioma stem-like cell (GSC) phenotype. It has been shown that CHRDL1 antagonizes the function of bone morphogenic protein 4 (BMP4), which induces GSC differentiation and, hence, reduces tumorigenicity. We, therefore, employed two previously described GSCs spheroid cultures and depleted them of CHRDL1 using the stable transduction of a CHRDL1-targeting shRNA. We show with in vitro cell-based assays (MTT, limiting dilution, and sphere formation assays), Western blots, irradiation procedures, and quantitative real-time PCR that the depletion of the secreted BMP4 antagonist CHRDL1 prominently decreases functional and molecular stemness traits resulting in enhanced radiation sensitivity. As a result, we postulate CHRDL1 as an enforcer of stemness in GSCs and find additional evidence that high CHRDL1 expression might also serve as a marker protein to determine BMP4 susceptibility.

Glioblastoma (GBM) is a grade IV primary brain tumor (WHO 2007). Despite last progresses in surgery, chemo- and radiotherapy, this brain tumor is still incurable and it always recurs (Stupp et al, 2009). It has been hypothesized that its resistance to therapies and its recurrence might be due to the existence of a subpopulation of tumor cells called Glioma Stem Cells (GSCs )(Galli et al, 2004; Bao et al, 2006). The goal of GBM therapy is the selective targeting of GSCs and, among the possibilities, the use of viral vectors has been widely investigated (Kroeger et al, 2010). Herpesviruses have been largely studied and modified in GBM treatment (Grandi et al, 2009). In this study the capacity of an engineered Bovine Herpesvirus type 4 (BoHV-4), named BoHV-4TKdsRED, to selectively kill GSCs has been investigated. The vector expresses the HSV-1 Thymidine Kinase and so it can be used with Ganciclovir (GCV) in a Suicide Gene Therapy protocol. Its potential role as a vector in GBM treatment has already been demonstrated (Redaelli et al, 2012). Three different primary cultures of human GSCs (GBM2, GBM4 and GBM5) and the corresponding serum-cultured cells (FBS2, FBS4 and FBS5) were used. They have been infected with BoHV-4TKdsRED and a combined treatment of the vector with GCV has also been tested on GSCs. It has also been analyzed whether signaling pathways of apoptosis or autophagy are activated in infected GSCs. To verify the BoHV-4TKdsRED effect on the cells counting/killing assays, plaque assays, Western Blot analysis and FACS analysis have been done. BoHV-4TKdsRED is able to infect GSCs and FBS cells. It can slightly replicate in almost all cultures examined and it can kill them in vitro. The combined treatment of the vector with GCV leads to an increased mortality in GSCs. Initial signs of autophagy activation were detected with the combined treatment in GSCs. Study of apoptosis through FACS analysis led to unreliable results. Another BoHV-4-derived vector, BoHV-4EGFPΔTK, demonstrated its ability in infecting selfrenewing cells, but without exerting a cytopathic effect. According to our results, BoHV-4TKdsRED would be a promising vector to target GSCs, but further analysis should be performed to strengthen these data.
Il Glioblastoma (GBM) è un tumore cerebrale primario di IV grado (WHO 2007). Nonostante gli ultimi progressi in chirurgia, chemioterapia e radioterapia, questo tumore al cervello è ancora incurabile e recidivante (Stupp et al, 2009). È stato ipotizzato che la sua resistenza alle terapie e le recidive potrebbero essere dovute all'esistenza di una sottopopolazione di cellule tumorali, denominata cellule staminali di glioma (Glioma Stem Cells - GSC) (Galli et al, 2004; Bao et al, 2006). L'obiettivo della terapia del GBM è quindi il targeting selettivo delle GSCs e, tra le possibilità, l'uso di vettori virali è stata ampiamente studiato (Kroeger et al, 2010). Gli Herpesvirus sono stati ampiamente studiati e modificati per il trattamento del GBM (Grandi et al, 2009). In questo studio è stata valutata la capacità di un vettore ingegnerizzato dall’ Herpesvirus Bovino di tipo 4 (BoHV-4), chiamato BoHV-4TKdsRED, di uccidere selettivamente le GSC. Il vettore esprime la Timidina Chinasi di HSV-1 e quindi può essere utilizzato con il Ganciclovir (GCV) in un protocollo di terapia genica suicida. Il suo potenziale ruolo come vettore per il trattamento del GBM è già stato dimostrato (Redaelli et al, 2012). Sono stati utilizzati tre diverse colture primarie di GSC umane (GBM2, GBM4 e GBM5) e le corrispondenti cellule coltivate in siero (FBS2, FBS4 e FBS5). Sono state infettate con BoHV- 4TKdsRED ed è stato testato anche un trattamento combinato del vettore con GCV sulle GSCs. È stato anche analizzato qualora vie di segnale di apoptosi o autofagia vengano attivate nelle GSCs infettate. Per verificare l'effetto di BoHV-4TKdsRED sono stati compiuti saggi di counting/killing, saggi di placca, Western Blot e analisi FACS. BoHV-4TKdsRED è in grado di infettare le GSCs e le cellule FBS. Può replicarsi in quasi tutte le colture esaminate e le può uccidere in vitro. Il trattamento combinato del vettore col GCV porta ad un aumento della mortalità nelle GSCs. Segni iniziali di attivazione dell'autofagia sono stati rilevati con il trattamento combinato nelle GSCs. Lo studio dell’apoptosi attraverso analisi FACS ha dato risultati non affidabili. Un altro vettore derivato da BoHV-4, BoHV-4EGFPΔTK, ha dimostrato la sua capacità di infettare le cellule self-renewing, ma senza esercitare un effetto citopatico. Secondo i nostri risultati, BoHV-4TKdsRED potrebbe essere un promettente vettore per le GSCs, ma ulteriori analisi devono essere effettuate per rafforzare questi dati.

Les gliomes sont les tumeurs cérébrales primitives les plus fréquentes de l’adulte. Le glioblastome (grade IV) en est la forme la plus agressive, caractérisé par sa résistance aux traitements actuels (chirurgie, chimiothérapie et radiothérapie). La mortalité de cette pathologie est quasi constante (survie médiane de 15 mois), ce qui justifie l’importance de découvrir de nouvelles cibles thérapeutiques. Le challenge est d'arriver à identifier des marqueurs spécifiques pour proposer un schéma thérapeutique alignant des stratégies de thérapies ciblées qui vont améliorer la prise en charge clinique, la survie globale et la survie sans progression des patients atteints de ces pathologies. Deux axes sont au centre des recherches fondamentales, translationnelles et cliniques. Le premier axe se définit autour du développement de molécules inhibitrices des voies de signalisation et le second autour du concept de cellules souches tumorales (CST) de glioblastomes (GBM) découvertes récemment dans le cerveau et qui révolutionnent la conception de la transformation tumorale.ASPM (Abnormal Spindle Like Microcéphaly Associated) est une cible candidate pertinente susceptible de participer au développement des gliomes (Horvath et al., 2007 ; Hagmann et al., 2008). Cette protéine régule la prolifération des neuroblastes, elle est fortement exprimée au stade embryonnaire, mais, reste faiblement exprimée dans le cerveau adulte. Par ailleurs, ASPM est impliquée dans divers processus de cancérisation (surexprimée dans les cancers du sein, du foie et du cerveau…), toute fois, le mécanisme responsable de cette dérégulation n’est pas encore bien caractérisé.Nos études menées sur une série de 169 gliomes humains, sélectionnés à partir de notre cohorte de patients, montrent que le gène ASPM est un marqueur de la progression vers la malignité, les grades les plus élevés exprimant le plus fortement ASPM. En outre, nous avons également montré que le niveau des transcrits d’ASPM est augmenté dans les récidives de gliomes et qu’en in vitro, ASPM contrôle la formation des gliomasphères (CST de GBM) avec une augmentation de l’expression de ses transcrits dans les cultures in vitro au fil des passages. En continuité de ces observations, nous avons alors développé un sh-miR-RNA spécifique d’ASPM permettant l’extinction post-transcriptionnelle de ce gène. Les résultats obtenus in vitro montrent que la perte d’expression d’ASPM conduit à un arrêt de la prolifération et aboutit à une mort cellulaire massive.Actuellement, des modèles de greffe de gliomasphères chez la souris (orthotopique) sont en cours de développement pour confirmer les effets observés in vitro et vérifier in vivo la validité de notre approche thérapeutique. En perspective, nous tenterons d’étudier les effets du silencing d’ASPM sur la voie de signalisation la plus dérégulée (pRB / E2F ou PI3K / AKT). Enfin, nous étudierons le rôle potentiel de cette protéine dans le contrôle du cycle cellulaire, et, in fine la mise en évidence de ses partenaires…
Glioblastoma (GBM) is the most frequent and aggressive form of primary brain tumors in adults; it is characterized by its resistance to current treatments (surgery, chemotherapy and radiotherapy). The prognosis is grim with a median survival of only 15 months underlining the importance to develop new therapeutic strategies. The recent development of the “tumor stem cell” (TSC) concept in hemopathies has been secondarily applied to gliomas with the identification of subpopulations of GBM cells which express neural stem cell markers and fulfill the criteria for stemness. Some evidences also suggest that this subpopulation could play a primary role in resistance to radio- and chemotherapy.ASPM (Abnormal Spindle Like Microcephaly Associated) is a protein regulating the proliferation of neuroblasts, highly expressed in the embryonic stage but weakly expressed in the adult brain. Preliminary reports suggesting that it could be involved in the development of gliomas (Horvath et al., 2007, Hagemann et al., 2008) prompted us to analyze further the role of this protein, focusing on its potential as a relevant candidate therapeutic target. In a series of 175 gliomas samples of various grades, we found that ASPM mRNA expression was strongly correlated with increasing tumor grade. We also found that ASPM expression increased at recurrence when compared to the initial lesion. Subsequently, we could demonstrate in vitro and in vivo that ASPM expression also increased over serial passages in gliomaspheres and in a mouse glioma xenograft model. In a therapeutic perspective, the effect of lentivirus-mediated shRNA post-transcriptional silencing of ASPM was evaluated in two different gliomasphere models and a dramatic proliferation arrest and cell death was observed. Taken together, these data suggest that ASPM is involved in the malignant progression of gliomas, possibly through expansion of a cancer stem cell compartment, and could be an attractive therapeutic target in glioblastoma multiforme.Another potential candidate tumor stem cell target in glioma is the sonic hedgehog pathway (hedgehog-Gli) which is required for GBM growth and stem cell expansion. In a collaborative study, it was found that NANOG, a transcription factor critically involved with self-renewal of undifferentiated embryonic stem cells, modulates gliomasphere clonogenicity, CD133+ stem cell behavior and proliferation. NANOG was regulated by hedgehog-Gli signalling and was essential for GBM tumourigenicity in orthotopic xenografts suggesting that it could also be a useful potential therapeutic target.Conclusions: Accumulating evidences suggest that tumor stem cells play an important role in the oncogenesis of gliomas and in their resistance to treatment. Our data support this concept and suggest that specific stemness markers may become useful targets to improve treatment of this devastating disease

Tese de doutoramento do Programa Interuniversitário de Doutoramento em Envelhecimento e Doenças Crónicas, apresentada à Faculdade de Medicina da Universidade de Coimbra
Glioblastoma (GBM) is the most malignant primary tumor of the central nervous system. Despite all efforts, the median survival time for GBM patients remains approximately between 12 to 15 months under therapy. GBM is a diffuse astrocytoma, highly proliferative, angiogenic, and locally invasive, that develops resistance to the alkylating agents used in chemotherapy, such as temozolomide (TMZ), which is considered part of the gold standard treatment. This limited success appears to be related with several mechanisms, namely: 1) the occurrence of gene mutations, that cause permanent activation and/or inhibition of several molecular signalling pathways involved in tumor growth and proliferation, such as protein kinase C (PKC) activation, cell survival, tumor suppressor genes and apoptosis; 2) the presence of a population of cells known to be chemo and radioresistant, the glioma stem-like cells (GSCs), that are responsible for generating tumor heterogeneity and recurrence after therapy, and; 3) the inexistence of a specific therapeutic target for non-GSCs and GSCs that would permit the development of more specific therapeutic approaches for this neoplasia. Therefore, in this work we aimed to: 1) study the PKC activation contribution to the aggressiveness of GBM, emphasizing the importance of combined therapeutic protocols, including TMZ with PKC inhibitors, namely tamoxifen (TMX); 2) characterize the GSCs and study their plasticity to understand glioma stem-like cells state and its differentiation properties, in order to contribute to the prevention of tumor recurrence; and 3) evaluate the potential of specific cell surface markers as therapeutic targets to non-GSCs and GSCs, allowing the accessibility of therapeutic agents most exclusively to the tumor niche, by a liposome-mediated drug delivery approach. First, using two GBM cell lines, the U87 and U118 cells, we observed that the combination of TMX and TMZ alters the phosphorylation status of PKC, by western blot. We found that TMX is an inhibitor of the p-PKC and that this combination is more effective in the reduction of proliferation and in the increase of apoptosis than each drug alone, by flow cytometry, which presents a new therapeutic strategy in GBM treatment. We then concluded that the combination of TMX and TMZ seems to potentiate the effect of each other in GBM cell lines. In order to study the heterogeneity between GBM cells and further understand the variability in the chemotherapeutic response, we next isolated and characterized a human GBM cell line, termed GBM11, obtained by surgical biopsy from a patient bearing a recurrent GBM, and compared the effect of TMX in monotherapy and in combination with TMZ on this GBM cell line with that observed in U87 and U118 cell lines. We observed that the effect of TMX plus TMZ or with TMX alone on GBM11 cells proliferation, death or migration capability, by flow cytometry and scratch assays, was similar, suggesting that, for recurrent tumors, the best choice of second-line treatment may be TMX alone, which may also reduce putative side effects of combined treatment with TMZ. The chemo- and radioresistance of GBM are also due to GSCs which contribute to tumor growth and relapse, highlighting this cell population as a main focus for GBM therapeutic research. We considered that the understanding of GBM stem state plasticity is of utmost importance to identify the mechanisms involved in GSCs resistance to therapy, which may justify tumor recurrence and so, constitute a step forward to the identification of new approaches to treat GBM. Our results demonstrated that, in four GBM cell lines and in the respectively GSC lines, the plasticity of the GBM stem-like cell state is based on the modulation of specific markers expression associated with this state, such as SOX2 or as Connexin 46 and 43, through immunofluorescence, western blot and PCR real time assays. Moreover, by immunohistochemistry analyses, we observed that this dynamic expression is in accordance with the upregulation of these stem-like cell markers in human samples of higher glioma grades, namely GBM, compared to lower grades, suggesting a direct correlation with the poor prognosis of GBM patients. As so, due to the plasticity of the stem-like cells status, the strategy of targeting both GSCs and non-GSCs may represent a promising approach in order to overcome tumor aggressiveness, and eventually to avoid the known chemotherapeutic side effects, which could improve the survival time and quality of life of GBM patients. In this regard, we next evaluate the potential of the cell surface nucleolin (NCL), described as overexpressed in cancer cells, as a target to specifically recognize non-GSCs and GSCs, taunting a possible therapeutic target for drug delivery in two different GBM cell lines. For that, we used a previously designed F3-peptide-targeted sterically stabilized pH-sensitive liposome (SLpH), which specifically recognizes nucleolin, as a tool to target overexpressed-nucleolin cells. Overall, we showed that NCL overexpression ensures an efficient drug delivery in both cells with stem-like and non-stem-like phenotypic characteristics, by flow cytometry assays, which could validate NCL as a potential therapeutic target in GBM. Altogether, our results showed: 1) a synergistic effect of TMX and TMZ in GBM cell lines and a more efficient effect of TMX alone in recurrent GBM compared to the combined therapy; 2) the plasticity of stem-like cell state through the reversibility of stem-like cell markers expression, and the identification of putative markers associated with this reversibility, the SOX2 and Cx46 and 43, which constitutes a step closer to the understanding of stem cell behaviour; and 3) that the success of targeting both non-GSCs and GSCs, through the nucleolin target, may be the basis for developing a specific treatment for GBM.
O Glioblastoma (GBM) é o tumor primário mais maligno do sistema nervoso central. Apesar de todos os esforços, o tempo médio de sobrevivência para doentes com GBM permanece aproximadamente entre os 12 a 15 meses sob terapia. O GBM é um astrocitoma difuso, altamente proliferativo, angiogénico e localmente invasivo, que desenvolve resistência aos agentes alquilantes utilizados na quimioterapia, como a temozolomida (TMZ), que é considerada parte do tratamento padrão. Este sucesso limitado parece estar relacionado com vários mecanismos, tais como: 1) a ocorrência de mutações genéticas que causam ativação permanente e / ou inibição de várias vias de sinalização molecular envolvidas no crescimento e proliferação de tumores, como a ativação da proteína cínase C (PKC), na sobrevivência celular, na inibição de genes supressores de tumores e apoptose; 2) a presença de uma população de células conhecidas como quimio- e radiorresistentes, as células de glioma do tipo estaminal (GSCs), que são responsáveis ​​pela heterogeneidade tumoral e recorrência após a terapia e; 3) a inexistência de um alvo terapêutico para não-GSCs e GSCs que permita o desenvolvimento de abordagens terapêuticas mais específicas para esta neoplasia. Assim, neste trabalho, objetivámos: 1) estudar a contribuição da ativação da PKC para a agressividade do GBM, enfatizando a importância de protocolos terapêuticos combinados, incluindo a TMZ com inibidores de PKC, nomeadamente o tamoxifeno (TMX); 2) caraterizar as GSCs e estudar a plasticidade das propriedades destas células estaminais do GBM, no sentido de compreender o estado estaminal do glioma e, consequentemente, entender as propriedades de diferenciação, contribuindo para a recorrência do tumor; e 3) avaliar o potencial de marcadores de superfície celular específicos, como alvos terapêuticos para as não-GSCs e GSCs, a fim de permitir a acessibilidade de agentes terapêuticos mais exclusivamente ao nicho do tumor, por meio de uma abordagem de administração de fármacos mediada por lipossomas. Inicialmente, usando duas linhas celulares de GBM, a U87 e a U118, observámos que a combinação de TMX e TMZ altera o estado de fosforilação da PKC, por western blot. Descobrimos que o TMX é um inibidor da p-PKC e que esta combinação é mais eficaz na redução da proliferação e no aumento da apoptose do que cada fármaco em monoterapia, através de ensaios de citometria de fluxo, o que pode representar uma nova estratégia terapêutica no tratamento do GBM. Concluímos, então, que a combinação de TMX e TMZ potencializa o efeito entre si nas linhas celulares de GBM. No sentido de estudar a heterogeneidade entre células de GBM e compreender melhor a variabilidade da resposta à quimioterapia, isolámos e caracterizámos uma linha celular de GBM humana, denominada GBM11, obtida através de uma biópsia cirúrgica de um doente com glioblastoma recorrente, e comparámos o efeito do TMX em monoterapia e em combinação com a TMZ, nesta linha celular, com o observado nas linhas celulares U87 e U118. Na verdade, observámos que o efeito do TMX e TMZ ou do TMX sozinho nas células de GBM11 sobre a proliferação celular, morte ou capacidade de migração, através de ensaios de citometria de fluxo e migração, era semelhante, o que pode sugerir que, para os tumores recorrentes, como o caso do GBM11 previamente tratado com TMZ, a melhor escolha do tratamento de segunda linha pode ser apenas TMX, a fim de reduzir os efeitos secundários putativos do tratamento combinado com TMZ. A quimio- e a radiorresistência do GBM devem-se, também, à existência de GSCs, que contribuem para o crescimento tumoral e recorrência destacando-se, assim, esta população celular como o foco principal da investigação terapêutica no GBM. Consideramos que a compreensão da plasticidade do estado estaminal no GBM é de extrema importância para identificar os mecanismos e fatores envolvidos na resistência das GSCs à terapia, o que pode justificar a recorrência do tumor e, portanto, constituir um progresso na identificação de novas abordagens terapêuticas. Os nossos resultados demonstraram, em quatro linhas celulares de GBM e nas respetivas linhas de GSCs, a plasticidade do estado estaminal com base na modulação da expressão de marcadores específicos associados, tais como o SOX2 e outros marcadores como a Conexina 46 e 43, através de ensaios de imunofluorescência, western blot e PCR em tempo real. Além disso, através de ensaios de imunohistoquímica, verificámos que essa expressão dinâmica está de acordo com a regulação positiva destes marcadores celulares em graus superiores de amostras humanas de glioma, nomeadamente no GBM, comparativamente a graus inferiores, sugerindo uma correlação direta com o mau prognóstico de doentes com GBM. Assim, devido à plasticidade do estado estaminal, a estratégia de atingir designadamente ambas as GSCs e não-GSCs pode representar uma abordagem importante no sentido de diminuir a agressividade do tumor e, eventualmente, evitar os efeitos colaterais quimioterapêuticos conhecidos, o que pode melhorar o tempo e a qualidade de vida de doentes com GBM. Neste sentido, avaliámos o potencial da nucleolina (NCL) de superfície celular, descrita como estando sobre-expressa nas células tumorais, como um alvo terapêutico para o reconhecimento específico de ambas as não-GSCs e GSCs, contribuindo para a entrega direcionada de fármacos encapsulados em nanopartículas, em duas linhas celulares de GBM. Para isso, utilizámos um lipossoma previamente desenhado, sensível ao pH e estericamente estabilizado, contendo na sua constituição um péptido F3, capaz de reconhecer especificamente a nucleolina constituindo, assim, uma ferramenta- alvo para as células com sobre-expressão de nucleolina. Em suma, demostrámos que a sobre-expressão de nucleolina per se pode identificar ambas as não-GSCs e GSCs, através de ensaios de citometria de fluxo, mediando a entrega direcionada intracelular, o que pode validar a NCL como um potencial alvo terapêutico no GBM. Em conclusão, o presente estudo demonstrou: 1) um efeito sinergístico do TMX e TMZ em linhas celulares de GBM e um efeito mais eficiente do TMX em monoterapia numa situação de GBM recorrente em comparação com a terapia combinada; 2) a plasticidade do estado estaminal através da reversibilidade da expressão dos marcadores de células do tipo estaminal e a identificação de dois marcadores putativos associados a essa reversibilidade, o SOX2 e a Cx46 e 43, constituindo um passo mais próximo na compreensão do comportamento das células estaminais; e 3) que o sucesso em atingir especificamente células não-GSCs e GSCs, através da sobre-expressão de nucleolina, poderá ser a base de desenvolvimento de um tratamento específico para o GBM.
Conselho Nacional de Desenvolvimento Tecnológico (CNPq), Brasil; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil; Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); Pró-Saúde - Associação Beneficente de Assistência Social e Hospitalar, Brasil; FEDER/COMPETE/ FCT PTDC/EBB-EBI/120634/2010 e PDTC/QUI-BIQ/120652/2010

The major roadblocks in treatment for GBM are resistance to therapy and recurrence of GBM cells. Regardless of various treatment strategies, the average survival of GBM patients is poor and incidence of recurrence remains high. The presence of GSCs, a dynamic cellular system within GBM contributes to chemo/radio resistance and recurrence. The plasticity of GSCs is supported by reversible biological processes including DNA methylation, histone modifications and RNA modifications. m6A is a reversible mRNA methylation which regulates various steps of RNA processing. In this thesis, we attempted to elucidate the METTL3- mediated m6A as a molecular mechanism behind the dynamic nature of GSCs. It comprises of two parts: Part 1- The landscape of METTL3-dependent m6A-epitranscriptome in GSCs and the functional orchestration of m6A targets Part 2- Essential role of METTL3 mediated m6A modification in glioma stem-like cells maintenance and radioresistance In first part, we demonstrate levels of m6A modified RNAs and METTL3 are maintained high in GSCs and they are attenuated during serum induced differentiation. Other members involved in m6A modification are either down regulated or unregulated in GSCs which confirms the dependency of GSCs in METTL3 for creating m6A marks. In addition, previous reports provide support that solely METTL3 carry catalytic domain for SAMbinding. Based on these facts, we elucidate the METTL3-dependent global m6A modification in GSCs and the impact of METTL3 targets in coordinating various functions. By comprehensive analysis of m6A-RIP seq and whole transcriptome post METTL3 silencing in GSCs, we identified the direct and indirect targets for METTL3-mediated m6A modification. The genes which preserve stem cell properties and aid in tumorigenesis were predominantly inhibited by METTL3 silencing in GSCs suggesting a universal oncogenic role of METTL3 in GBM. Large subset of genes was down regulated after METTL3 silencing suggesting a global destabilization of transcripts. The enrichment of m6A peaks near stop codon and 3’UTR indicates functional importance of METTL3 in RNA stabilization and translation termination. In addition to protein coding genes, METTL3 fine-tunes the expression of non coding RNAs which includes lncRNAs, anti-sense RNAs etc. Interestingly, we identified m6A peaks which encompass miRNA target sites and we hypothesize that m6A modification may scrutinize the binding affinity of miRNAs. We further determined the inter-play between chromatin remodeling with m6A epitranscriptome. Genes which carry active chromatin marks and transcription factor binding are further stabilized by METLT3- dependent m6A modification. The altered secondary/tertiary structure induced by m6A may act as loading site for various RBPs and achieve various RNA processing functions. Our analysis deduced RBPs HuR and QKI preferentially bind to m6A marked RNAs and helps in enhancing the expression of m6A modified targets. Together, this study provides a panoramic view on global m6A modification mediated by METTL3 in GSCs. In second part, we examined the crucial role of METTL3 in glioma stem cell physiology. Inhibition of METTL3 hinder the neurosphere formation and stem cell properties of GSCs. Anti-METTL3 RIP studies combined with m6A RIP-seq results identified SOX2 as a key m6A mediator of METTL3 and the m6A marks created by METTL3 sustains SOX2 transcript stability. The exogenous over expression of 3’UTR-less SOX2 significantly alleviated the inhibition of neurosphere formation observed in METTL3 silenced GSCs. METTL3 interaction and m6A modification in vivo required intact three METTL3/m6A sites present in the SOX2- 3’UTR. Further, we found that HuR recruitment to m6A modified RNA is essential for SOX2 mRNA stabilization by METTL3 and at global level HuR-RNA interaction prefers the m6A modified transcripts. METTL3 silenced GSCs showed enhanced sensitivity to γ-irradiation due to reduced DNA repair. Exogenous overexpression of 3’UTRless SOX2 in METTL3 silenced GSCs rescues efficiency of DNA repair and specifically homologous recombination repair. It also resulted in the significant rescue of neurosphere formation from METTL3 silencing induced radiosensitivity. GBM tumors have elevated METTL3 transcripts and silencing METTL3 in GSCs inhibited tumor growth and prolonged mice survival. METTL3 transcript levels predicted poor survival in GBMs which are enriched for GSC-specific signature. Thus our study reports the importance of m6A modification in GSCs and uncovers METTL3 as a potential molecular target in GBM therapy

The central nervous system tissue contains neural stem cells which differentiate and give rise to the neurons and glial cells. The glial cells maintain neurons by providing nutrients, physical support and protection. Unlike neurons, glial cells retain their capacity to divide and proliferate throughout the life span of an individual. During proliferation of glial cells, dysregulation in cellular processes that control growth leads to formation of glial neoplasms or gliomas. The glial tumors are classified based on the predominant cell type; astrocytomas that originate mainly from astrocytes, oligodendrogliomas from oligodendrocytes and oligoastrocytomas both astrocytes and oligodendrocytes. Among all gliomas, astrocytomas are the most common central nervous system neoplasms which makes up to 60% of all the primary brain tumors. Being the most prevalent type, the world health organizations (WHO) classifies them into grades ranging from I to IV based on their intensity of malignancy. Grade-IV astrocytoma or Glioblastoma (GBM) is the most malignant form with a median survival of less than 15 months, despite all therapeutic modalities. CAMs are involved in cell-cell and cell-extra cellular matrix (ECM) communications and physical attachment between cells and ECM. CAMs are also involved in ‘out-in and in-out’ signalling which helps in cell survival, migration and stemness in both normal and cancer cells. Cell adhesion system works in various mechanisms by which it translates basic genetic information into complex three-dimensional pattern of cells in tissues. Dysregulation of CAMs is involved in different neoplastic transformation, survival, self-renewal, proliferation, migration, EMT, and metastasis. The aim of this study was to investigate the role of CAMs in glioma development and progression. Part 1: Elucidation of Genetic and Epigenetic landscape alterations in Cell Adhesion Molecules (CAMs) in Glioblastoma CAMs are important group of molecules expressed on the surface of the cell and often found dysregulated in multiple cancers including GBM. Though several reports emphasize the importance of a number of individual CAMs in glioma initiation and progression, there is no single report which shows the comprehensive view of their importance in glioma development and progression. In this study, we have curated all known CAMs from the literature and analysed using various bioinformatics tools to show their genetic alterations, probable mechanisms for regulation, association with tumor grade/nature, and survival correlation in GBM. A comprehensive bioinformatics analysis of CAMs (n=518) in multiple available data sets revealed genetic and epigenetic alterations among CAMs in GBM. We identified several genetically altered CAMs (n=293, 56%) in GBM. The mutations in the LRFN5 and PCDHGC5 predicted poor prognosis. The differential regulation of CAMs (n=181, 35%) contributed by copy number variation (CNV), DNA methylation and miRNA targeting. We identified two sets of differentially regulated CAMs that may be implicated in initial astrocytic transformation and glioma progression. Further, we have also identified a unique set of differentially regulated CAMs (n=22) specific to glioma stem-like cells (GSC) as compared to both neural stem cells (NSC) and differentiated glioma cells (DGC). Part 2: The essential role of Prostaglandin F2 receptor inhibitor (PTGFRN) in glioblastoma PTGFRN is an IgSF CAM, surface expressed single pass transmembrane protein and serves as a scaffolding protein. It is widely found to be associated with multiple tetraspanin members such as CD9, CD81, CD82 and CD151. It was reported as a highly expressed antigen in metastatic cancer cells and induces cell migration in multiple cancer types. In our study, integrative analysis of CAMs revealed that PTGFRN is one of the upregulated genes in GSCs compared to both DGCs and NSCs. In this section, we identified PTGFRN to be highly expressed in GSCs vs DGCs, GBM samples compared to controls as analysed in publicly available multiple datasets such as TCGA (Agilent and RNA-seq), REMBRANDT, GSE22866 and GSE7696. Further, when we explored the expression of PTGFRN in GBM cell lines, transcript and protein levels were found to be more in GBM cell lines as compared to immortalized astrocyte derived control cell lines. The survival analysis revealed that PTGFRN high expression is correlated with poor patient survival in GBM. The functional relevance of PTGFRN in GBM development and progression was investigated by shRNA mediated silencing followed by functional assays. In multiple GBM cell lines, silencing of PTGFRN reduced cell proliferation, colony formation and anchorage-independent growth in soft agar as compared to silencing of non-targeting (shNT) control. Silencing of PTGFRN in GSCs reduced number of neurospheres formed as compared to shNT control as assessed by neurosphere formation and limiting dilution assays. We also found that the knockdown of PTGFRN reduces migration and invasion in GBM cell lines. Cell cycle analysis showed G1 or G2/M phase arrest after silencing PTGFRN in GBM cell lines. Further, we observed that in GBM cell lines silencing of PTGFRN leads to apoptotic cell death by 40 to 50% as compared to shNT control. In order to understand the functional role of PTGFRN in GBM, we first identified the significantly differentially regulated genes between PTGFRN-high and PTGFRN-low expressed groups of GBM samples in TCGA Agilent dataset. The gene sets were further used for KEGG pathway, Gene Ontology (GO) and GSEA analyses. We found that the upregulated genes showed significant enrichment for focal adhesion and CAMs pathways, whereas negatively regulated genes showed significant enrichment for drug and xenobiotic metabolism by cytochrome p450 pathways in KEGG pathway database. The GO and GSEA analyses showed significant enrichment for hallmarks: Epithelial-mesenchymal transition (EMT), IL6 JAK STAT3 signalling, TNF alpha signalling via NF-kB and Notch signalling and significantly depleted for the hallmark: KRAS signalling DN (genes down-regulated by KRAS activation). To decipher the molecular mechanism for PTGFRN function in GBM, we checked the status of the most dysregulated oncogenic pathways after its knockdown in GBM cell lines. Silencing of PTGFN reduced phospho-AKT, phospho-ERK, phospho-4EBP and phospho-P70S6 levels compared to control condition in GBM cell lines. In order to test the role of PTGFRN in EMT and Focal adhesion processes, we silenced PTGFRN in GBM cell lines and observed a reduction in vimentin and Focal Adhesion Kinase (FAK) protein levels compared to control. It indicates that PTGFRN might be playing a potential role in pro-survival and promigratory pathways in GBM. Furthermore, we also evaluated various mechanisms by which PTGFRN expression is regulated in GBM. Bioinformatics analysis revealed that DNA hypomethylation and down regulation of microRNAs such as miR-107, miR-133b and miR-137 correlated with PTGFRN high expression in GBM. In order to test the role of methylation, we treated LN18 cell line (PTGFRN-low) with 5-Aza 2’-cytidine and observed that no rescue of PTGFRN expression which indicates that methylation may not play a role in its regulation. However, overexpression of miR-137 in the PTGFRN-high glioma cell lines (U373, U251 and U87) reduced PTGFRN protein levels indicating microRNA-dependent regulation of PTGFRN. Moreover, a recent report suggests that TGF-β signalling pathway may also regulate PTGFRN in GBM. Inhibition of TGF-β signalling with ALK5 inhibitor also reduced PTGFRN protein levels relative to the control treatment. The high activation of TGF-β signalling and down regulation of miR-137 expression might be responsible for high expression of PTGFRN in GBM. Part 3: Astrotactin 1 (ASTN1), a neural CAM is essential for survival and growth of Glioma Stem-like Cells Astrotactins (ASTN1 and ASTN2) are neural CAMs which are known to be involved in the development of CNS. ASTN1 was also shown to be upregulated in premalignant stem-like or progenitor cells of glioblastoma compared to astrocytes and hESCs. In our study, integrated gene expression analysis revealed ASTN1 as a highly upregulated CAM in GSCs compared to both NSCs and DGCs. We analysed ASTN1 transcript and protein levels in various GSC cell lines and corresponding differentiated glioma cells. We found ASTN1 to be upregulated in GSCs compared to corresponding DGCs at both transcript and protein levels. Silencing of ASTN1 in GSCs formed reduced number of neurospheres in neurosphere and limiting dilution assays as compared to shNT control. We also found that the cell viability was significantly reduced after silencing of ASTN1 in GSCs as assessed by trypan blue exclusion assay. In addition to knockdown, blocking of ASTN1 with antibody also inhibited the neurosphere formation in GSCs. We further investigated the mechanisms by which ASTN1 exhibits its role in GSC survival and growth. Silencing of ASTN1 in GSCs increased the number of annexin-V positive cells which indicates increased apoptosis. To further validate ASTN1 role in GSC stemness maintenance, we checked the essential four-reprogramming factors; Sox2, Po3f2, Olig2, and Sall2 which are necessary and sufficient for maintenance of stemness in GSCs. In GSCs, silencing of ASTN1 reduced transcript levels of all four reprogramming factors as compared to vector control. In conclusion, we have elucidated the altered landscape of CAMs in GBM and given an insight into impact of such alterations on functional and molecular role taking PTGFRN and ASTN1 as examples. In first part we provided a panoramic view of the various alterations in CAMs encountered in GBM. In second part, we identified that PTGFRN is required for GBM cell growth, migration, and invasion and we also found its regulation. In the third part, we demonstrated that ASTN1 is required for GSC survival and growth.

Gliomas are primary brain tumors in adults that are believed to originate from different types of glial cells. Central nervous system gliomas pose particularly difficult problems because of their tendency towards malignancy, rate of tumor spread, and the lack of effective therapy. Glioblastoma (GBM/ grade IV glioma) is one of the most aggressive types of gliomas. Despite the advancement in treatment modalities including surgery, chemotherapy and radiotherapy, the overall median survival of a GBM patient remains at 14.6 months. This dismal overall picture of survival is further aggravated by the grim quality of life of the GBM patients. Advances in high-throughput technologies have enabled us to mine the molecular pathways contributing to pathogenesis and resistance of GBM. Integrated genomic and epigenomic screens have revealed several molecular markers and has tremendously improved the classification of these heterogeneous tumors. These finding have also given key insights into the various genetic and epigenetic derailments that contribute towards gliomagenesis. Broadly speaking, genomics and transcriptomics for several decades has kept the focus of research on mutation, copy number variation, chromatin remodellers and transcriptional regulation as major determinants of gene expression alterations in cancer. Recent advances in these fields have paved the way to gain a better understanding of the regulation imposed at the post-transcriptional level. Non-coding RNAs and RNA binding proteins (RBPs) are emerging as important post-transcriptional modulators of gene expression. Our study illustrates the various aspects of RBP biology in GBM, where our focus ranges from surfacing the RBP landscape of GBM, delineating the magnitude of regulation of a particular RBP, IMP3 (IGF2 mRNA binding protein 3) and also determine one of the targets of this protein which contributes towards glioma stem-like cell (GSC) maintenance in GBM. The thesis is divided in three work-related chapters: chapter 3, chapter 4 and chapter 5. In chapter 3, we have performed extensive bioinformatics analyses to get an idea about the altered RBPome of GBM and the probable mechanisms leading to this mis-regulation. In chapter 4, we exemplify IMP3 to study the extent of multi-level post-transcriptional regulation imposed by RBPs in glioma cells. Further, in chapter 5, we conclude that IMP3 is a critical RBP for GSC maintenance. Moreover, we establish p65, a subunit of NF-κB pathway as a translationally activated target of IMP3. p65 also acts as a downstream mediator of IMP3 in maintaining glioma stem-like cell survival and proliferation. CHAPTER 3: Elucidation of the genetic and epigenetic landscape alterations in RNA binding proteins in glioblastoma RNA binding proteins (RBPs) are global regulators which participate in various steps of RNA metabolism. Though, recent studies have indicated their importance in diseases including cancer, the molecular mechanisms regulated by these proteins still remains elusive. In this study, we have carried out an integrated bioinformatics analysis of the status of RBPs (n = 1756) in various datasets (n = 11) to identify several genetic and epigenetically altered events among RBPs in GBM. Alterations in RBPs which included mutation, InDels (Insertion and Deletions) and expression level changes, which may lead to aberrant activity of these global regulators were assessed in our analyses. Whole exome sequencing (WES) data from The Cancer Genome Atlas (TCGA) was analysed to identify mutated RBPs in GBM. We identified 13 RBPs to be mutated in minimum of 2% of GBM samples. Further, correlation of mutations in AHNAK predicted poor prognosis. Integrated analyses of transcriptome, Copy Number Variation (CNV) data, DNA methylome and miRnome were carried out to identify differentially regulated RBPs. There were 472 differentially regulated RBPs. Additional analysis revealed that a significant proportion of differential regulation is due to CNV (9%), DNA methylation (9.5%) and miRNA targeting (37%). To obtain insights into transformation and aggressiveness related RBPs in GBM, we analysed transcript levels of RBPs in grade II and grade IV (GBM) samples. Differentially regulated RBPs in grade II astrocytoma when compared to control brain samples, which had similar expression in GBM were identified as transformation related RBPs. On the other hand, RBPs that showed differential regulation only in GBM when compared to grade II and control brain samples were identified as aggressiveness related RBPs. These two sets of genes may be implicated in initial astrocytic transformation and glioma progression respectively. We also compared the transcriptome data of neural stem cells (NSC), glioma stem-like cells (GSC) and differentiated glioma cells (DGC) to identify GSC specific RBPs signature, which was further subjected to survival analysis and gene set enrichment analysis. These analyses led to the identification of a unique set of differentially regulated RBPs (n = 34) specifically in GSCs compared to NSC and DGC. RBP risk score derived from four prognostic RBPs (NOL3, SUCLG1, HERC5 and AFF3) is demonstrated to be an independent poor prognostic indicator in GBM. RBP risk score also stratified GBM patients into low-risk and high-risk groups with a significant survival difference. Gene set enrichment analysis (GSEA) of differentially regulated genes between high-risk and low-risk identified positive enrichment of NF-κB, inflammatory response, epithelial mesenchymal transition and hypoxia pathways in high-risk GBM. Thus, our study provides a comprehensive overview of genetic and epigenetic regulation of RBPs in glioma development and progression. CHAPTER 4: Transcriptome and translatome regulation by IMP3 in glioma cells IMP3 in the previous section was found to be an upregulated and aggressiveness related RBP in GBM. Previous work from our laboratory had established IMP3 as an RBP which contributes to proliferation, migration, invasion, chemoresistance and angiogenesis of glioma cell lines. Moreover, it was shown to enhance IGF2 translation, without altering its transcript levels. This regulation was attributed to the increased activation of pro-survival pathways like PI3K and MAPK in IMP3 overexpressing cells. Enormous volume of literature demonstrates that RBPs are bestowed with the ability to bind multiple targets and regulate their fate at various levels including their degradation, localization and translation. Recent research has laid emphasis on RBPs as global regulators of RNA metabolism and translation. Hence, we were intrigued to understand the effect of IMP3 on transcriptome and translatome of glioma cells. To get insights in IMP3 modulated transcriptome, we performed a microarray based global gene profiling of total cellular RNA of IMP3 silenced U251 cells. We identified 2788 differentially regulated genes at the transcript level. Further, these differentially regulated genes were classified as direct and indirect targets depending on the presence of IMP3 binding sites. We speculate that the RNA stability of these direct targets may be regulated by IMP3. Our observations suggest that, IMP3 may act as a bimodular regulator of RNA stability of the identified direct targets. This implies that few of the targets may be stabilized while others may be destabilized by IMP3 binding. Correlation of the expression of the differentially regulated targets with IMP3 transcript in GBM tumors (TCGA data is used) revealed a list of genes which is more likely to be regulated by IMP3 in GBM tumors. Biological processes enrichment analysis of these direct and correlated targets suggested that IMP3 regulates cell cycle progression by regulating these genes. Next, we were interested in identifying the genes getting differentially regulated exclusively at the level of translation. Polysome analysis was performed on IMP3 silenced cells, and RNA from the pooled heavy polysome fractions was subjected to microarray. Differentially regulated genes in heavy polysome fractions whose expression remain unaltered in total RNA were selected for further investigation. Similar trend of bimodular regulation was also observed at the level of translation. Direct and indirect targets were identified on the basis of presence of IMP3 binding sites. Interestingly, we found that several direct translation targets of IMP3 were associated with apoptosis and cell death related pathways. We have also directed our efforts in unravelling few of the possible mechanisms which may contribute in regulating the indirect targets, at transcriptome and translatome level. Transcription factors (TFs) and RBPs which are direct targets of IMP3, and which may influence the gene expression of indirect targets of IMP3 were identified. Taken together, we have unravelled the IMP3 regulons in glioma cells and their role in cell cycle progression and apoptosis. CHAPTER 5: IMP3 contributes to glioma stem-like cell maintenance and chemoresistance by promoting the translation of RelA/p65 Therapy resistance presents a severe challenge in battling GBM. One of the culprits for chemo- and radioresistance identified in GBM tumors is a small proportion of slow-dividing glioma stem-like cells (GSCs) which are refractory to current treatment modalities. These cells are thus spared by the treatment and then repopulate to give rise to an even more belligerent recurrent tumor. Identification of molecules specifically expressed in GSCs, but not in their normal counterparts (adult human neural stem cells-ahNSC) may provide potential lucrative targets for therapy. Interestingly, using a publically available microarray data for GSC and ahNSC, we found that IMP3 was the most upregulated RBP in GSCs as compared to ahNSC. Our Gene Set Enrichment Analysis results indicated that GSC signature genes were also closely linked to IMP3 expression in GBM tumors. Experiments carried out in IMP3 silenced conditions revealed that IMP3 is required for GSC maintenance and imparts chemoresistance to glioma cells and GSCs. These experiments provide compelling evidence that expression of IMP3 is imperative for GSC survival and their chemoresistance. Owing to the promiscuous binding of RNA binding proteins, we were intrigued to identify a direct target regulated by IMP3 which is also necessary for GSC maintenance by IMP3. We were keen to identify the transcription factors which are regulated by IMP3 at the translation level. Thus, we focussed on the transcription factors harbouring IMP3 binding sites and which may be unregulated at the transcript level by IMP3. Integrated bioinformatic analysis using published datasets revealed RELA (p65) as a potential target which fulfilled all the aforementioned criteria. Ectopic overexpression and silencing of IMP3 in glioma cell lines confirmed that IMP3 increases the NF-κB pathway activity. Moreover, as expected, p65 transcript levels did not change under IMP3 modulated conditions, while significant protein level changes were observed. IMP3 overexpression led to increased p65 protein levels, while its silencing reduced p65 protein levels significantly in the glioma cell lines. Furthermore, the reduced concentration of p65 protein in IMP3 depleted cells was due to decreased translation of p65 transcript in these conditions, with no effect on its protein stability upon IMP3 modulation. We also establish that IMP3 directly binds to three sites present at the 3’UTR of p65 transcript. Furthermore, alleviation of decrease in neurosphere numbers was observed upon exogenous overexpression of p65 in IMP3 silenced GSCs. We also establish IMP3 as a transcriptional target of p65. Taken together, this study establishes p65 as a novel and bonafide target of IMP3 and as a mediator of IMP3 in GSC maintenance. It also underscores the significance of IMP3 as a therapeutic target, which can indirectly be used to target NF-κB pathway in glioblastoma.

Glioblastoma multiforme is one of the most aggressive tumors of the central nervous system. The current standard-of-care includes maximal resection followed by chemotherapy, radiation and more recently, tumor treating fields (TTFs). Despite this multimodal approach, glioblastoma remains refractory to therapy. Glioblastoma resistance, recurrence and malignancy are believed to be driven by a subpopulation of glioma stem cells (GSCs) within the tumor bulk which are characterized by the retention of self-renewal potential as well as the capacity to recapitulate tumor heterogeneity. Within the dynamic intratumoral niche, GSCs demonstrate a high degree of cellular plasticity, reversibly interconverting between stem-like states and more differentiated states as a result of environmental cues/signaling fluctuations. Such plastic adaptive properties are mostly driven by multiple dynamic, reversible epigenetic modifications. We posit that reversible post-transcriptional methylation of RNA transcripts at the m6A position may be one such regulatory mechanism employed by GSCs to efficiently maintain plasticity and adaptive phenotypic transitions. In this section, we discuss the concept of cellular plasticity, introduce dynamic m6a epitranscriptomic mechanisms as potential key regulators of GSC plasticity and finally propose epigenetic based therapeutics as a mean of attenuating glioblastoma plasticity to improve patient outcome.