Existing methods of treating glioma are not effective for eradicating the disease. Therefore, new and innovative methods of treatment alone or in combination with existing therapies are necessary. Delivery of therapeutic agents through delivery carriers such as liposomes diminishes the harmful effects of the agent in healthy tissues and allows increased accumulation in the tumor. In addition, targeted chemotherapy using liposomes provides the opportunity for further increase in drug accumulation in tumor. However, the current targeting strategies suffer accelerated plasma clearance and are not advantageous in improving efficacy. The search for new tumor targets, novel ligands, new strategies for targeting, and particle stabilization will advance our ability to improve delivery at the tumor level while decreasing toxicity to normal tissues. The global objective of this thesis was to improve the status of current liposomal therapy to achieve higher efficacy in tumors. Here, we show a novel mechanism to increase targeting to tumor while uncompromising on the long circulation of stealth liposomes. Long circulation is essential for passive accumulation of the nanocarriers due to EPR effect, in order to see benefits of targeting. Using phage display technique, a variety of tumor specific peptides were identified for use as targeting moieties. One potential advantage of the approach proposed here is the rapid identification of patient tumor specific peptide that evades the RES. This could lead to the development of a nanocarrier system with high avidity and selectivity for tumors. Therefore, tumor accumulation of the targeted formulations will be higher than that of non‐targeted liposomes due to increased drug retention at the tumor site and uncompromised blood residence time.In addition, it has been shown that the distribution of nanocarriers, spatially within the tumor, is limited that might further hinder the distribution of the encapsulated drug, thereby limiting efficacy. It is necessary to release the drug from within the nanocarrier to promote increased efficacy. Here, we were able to address the problem of drug diffusion within the tumor interstitium using a combination therapy employing a remotely triggered thermosensitive liposomal chemotherapeutic. We fabricated a thermosensitive liposomal nanocarrier that maintained its stability at physiological temperature to minimize toxicity to healthy cells. We, then, showed a remote triggering mechanism mediated by gold nanorods heated via NIR can help in achieving precise control over the desired site for drug release. These strategies enabled increased drug availability at the tumor site and contributed to tumor retardation. Additionally, we show that the synergistic therapy employing gold nanorods and thermosensitive liposomes may have great potential to be translated to the clinic.

Since Parsons et al. (2008) found the frequent mutations of IDH1 (12%) in GBMs, various reports have studied the prevalence and characteristic of IDH1 and IDH2 mutations.The mutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in nearly 40% of gliomas. The frequency of IDH1 mutations are inversely connected with grade II (~80%), III (~50%), and IV (~ 10%) gliomas. Importantly, the status of IDH1 mutations is associated with a better outcome and demonstrated a diagnostic value. We analyzed also these mutations in distribution, association with tumor-derived other genetic alterations and the diagnostic and prognostic value in a cohort of 1332 glioma patients.A synonymous single nucleotide polymorphism [SNP rs 11554137; C (cytosine) substituted by T (thymin)] has been studied in gliomas patients. The SNP rs 11554137 (in codon 105) are located in the same exon with the IDH1 R132 mutations (in codon 132). And gliomas patients with SNP rs 11554137: C>T had a poorer outcome than patients without SNP rs 11554137. This was observed a similarly adverse effect in survival in patients with AML. Mutations in codon 132 can cause a decrease of IDH1/2 activity and also gain a new enzyme function for the NADPH dependent reduction of alpha-ketoglutarate to 2-hydroxyglutarate. High 2HG and low NADPH levels might sensitize tumors to oxidative stress, potentiating response to radiotherapy, and may account for the prolonged survival of patients harboring the mutations. So we studied further the alterations of function in IDH1R132H mutant cells in vitro. Based on the decrease of defence and the increase of impairing factors in tumor cells, we found that the tumors harbouring IDH1 mutations may have an elevated radiosensitivity. In the present study, we described the impact of IDH1 mutations in gliomas and search for new perspectives for the treatment strategy.

Le glioblastome est la plus fréquente et la plus agressive des tumeurs cérébrales primaires.<p>Malgré un traitement standard pluridisciplinaire (chirurgie, radiothérapie et chimiothérapie),<p>la médiane de survie des patients atteints de ce type de tumeur est de 15 mois et aucun patient<p>n’a pu être guéri à ce jour. Ce pronostic très sombre est directement lié à la capacité<p>d’invasion diffuse du parenchyme cérébral par les cellules gliales tumorales, ce qui rend<p>impossible une résection chirurgicale totale. De plus, ces cellules sont particulièrement<p>résistantes aux traitements chimiothérapeutiques de type pro-apoptotique, entrainant une<p>récidive quasi inévitable de la tumeur. De nombreuses stratégies thérapeutiques telles que<p>l’immunothérapie ou les thérapies ciblées sont actuellement explorées pour tenter de<p>combattre ces tumeurs. Cependant, leur efficacité au niveau clinique s’est avérée décevante. Il<p>devient donc indispensable d’identifier de nouveaux agents thérapeutiques afin d’améliorer la<p>survie des patients atteints de glioblastome.<p>Dans ce travail, nous avons caractérisé les propriétés anticancéreuses in vitro et in vivo de<p>deux terpénoïdes extraits de champignons, la fusicoccine A et l’ophioboline A, puis nous<p>avons caractérisé en partie leur mécanisme d’action anti-tumoral dans des cellules de<p>glioblastome.<p>Tout d’abord, nous avons montré que l’activité inhibitrice de croissance in vitro de la<p>fusicoccine A et de l’ophioboline A n’était pas dépendante du degré de sensibilité ou de<p>résistance à l’apoptose des cellules gliales tumorales. Nous avons ensuite mis en évidence que<p>la fusicoccine A était capable de diminuer l’invasion des cellules de glioblastome in vitro en<p>ciblant la kinase focale d’adhérence (FAK). Dans le même temps, nous avons démontré que<p>l’ophioboline A était capable d’induire la mort de ces cellules par paraptose en inhibant<p>l’activité du canal ionique BKCa. Ces deux cibles sont intéressantes car en plus d’être<p>surexprimées dans les glioblastomes, elles interviennent dans de nombreux processus<p>cellulaires tels que la prolifération, la migration et la survie cellulaire.<p>Pour finir, nous avons analysé le pouvoir anti-tumoral in vivo de ces deux terpénoïdes en<p>utilisant un modèle murin de mélanome métastatique, couramment utilisé dans notre<p>laboratoire. Seule l’ophioboline A, injectée en intrapéritonéal à 10 mg/kg, augmentait de<p>manière significative la survie des souris traitées avec cette molécule par rapport aux souris<p>contrôles. Dans ce premier modèle, nous n’avions pas déterminé les conditions optimales<p>9<p>pour l’évaluation in vivo de la fusicoccine A et de l’ophioboline A. Lorsque celles-ci seront<p>définies, des modèles de xénogreffes orthotopiques de glioblastomes humains implantées dans<p>le cerveau de souris immunodéficientes seront utilisés.<p>En conclusion, l’ophioboline A, pouvant être produite en quantités industrielles par culture<p>du champignon qui la synthétise, possédant un mécanisme d’action original et montrant déjà<p>un début d’activité in vivo, pourrait représenter une molécule méritant des études plus<p>approfondies en termes d’agent thérapeutique susceptible de combattre les glioblastomes. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Les gliomes malins sont caractérisés par une prolifération importante, une migration diffuse des astrocytes tumoraux dans le parenchyme cérébral et un taux important de néo-angiogenèse. La gastrine appartient à la famille des peptides apparentés à la cholécystokinine et cette dernière est présente en abondance dans le cerveau. De plus la gastrine est capable de modifier le comportement biologique d’un certain nombre de tumeurs. Le groupe de recherche au sein duquel j’ai réalisé mon travail de thèse fut le premier à suggérer le rôle potentiel de la gastrine au niveau des taux de prolifération et de migration des astrocytes tumoraux. Nous avons précisé dans le présent travail divers effets biologiques provoqués par la gastrine au niveau de gliomes et de gliosarcome expérimentaux.<p>Nous avons au préalable tenté de caractériser par une technique de RT-PCR l’expression d’ARN pour divers récepteurs à la gastrine au sein de tumeurs du système nerveux central et périphérique (comprenant des gliomes, des méningiomes et des schwannomes), au sein de gliomes et d’un gliosarcome expérimentaux, et au sein de cellules endothéliales humaines de veines ombilicales HUVEC et de manchons vasculaires obtenus par microdissection au laser d’un glioblastome humain. Nous avons également développé un modèle de neurochirurgie expérimentale chez le rat consistant en la résection microchirurgicale de la tumeur cérébrale après un bilan iconographique par IRM. Nous avons ainsi montré que l’administration de gastrine dans le foyer opératoire après résection tumorale augmente significativement la période de survie de rats immunodéficients porteurs du modèle de gliome humain U373 et de rats conventionnels porteurs du modèle C6 de rat. In vitro, nous avons montré grâce au test colorimétrique MTT que la gastrine induit une diminution significative du taux global de croissance de ces deux modèles avec une accumulation des astrocytes tumoraux dans la phase G1 de leur cycle cellulaire. Par la technique de Western blotting nous avons également montré que la gastrine induit une diminution significative des taux protéiques du complexe cycline D3-Cdk4 dans les deux modèles expérimentaux. Nous avons montré que la gastrine est capable de réduire significativement l’invasion des modèles C6 de rat, U373 humain et de gliosarcome 9L de rat au travers d’une matrice de collagène et de réduire l’invasion des cellules U373 en chambre de Boyden. La gastrine modifie également significativement la motilité des cellules C6 et U373 et l’organisation de leur cytosquelette d’actine.<p>Nous avons découvert que la gastrine administrée en intracérébral dans le foyer tumoral U373 augmente significativement le taux d’angiogenèse au sein de la tumeur. Nous avons alors investigué l’effet de la gastrine et des antagonistes des récepteurs à cholécystokinine sur le taux d’angiogenèse in vitro en utilisant le modèle des cellules HUVEC cultivées sur Matrigel. L’effet pro-angiogénique in vitro et in vivo de la gastrine est significativement contrecarré par le produit L365,260, un antagoniste relativement spécifique du récepteur CCK-B de la gastrine. La gastrine est chémoattractante sur les cellules HUVEC et augmente significativement leur sécrétion d’IL-8. Toutefois l’effet pro-angiogénique de la gastrine serait en partie dépendant de la modification du taux d’expression des sélectines par les cellules HUVEC, et non de la sécrétion d’IL-8. Nous avons réalisé une revue de la littérature pour tenter de comprendre pourquoi les astrocytes tumoraux migrants sont résistants à la chimiothérapie conventionnelle. A la fin du chapitre Discussion, dans le sous-chapitre intitulé « Quels sont les espoirs thérapeutiques dans le cas des gliomes dits diffus? », nous tentons d’analyser les implications thérapeutiques potentielles qu’il serait possible de tirer du présent travail. <p><p><p> / Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

Gliomas are the most common primary brain tumors and include astrocytomas, oligodendrogliomas and oligoastrocytomas. Astrocytomas have a high frequency of occurrence as compared to the other gliomas and several studies including ours have focused on understanding the etiology, biology and genetics of this disease. Based on the degree of malignancy, astrocytomas have been graded from I to IV. Grade I or pilocytic astrocytomas are benign tumors and have limited infiltration. On the contrary, Grade II-IV astrocytomas also referred to as diffusely infiltrating astrocytomas (DA, Grade II), anaplastic astrocytomas (AA, Grade III) and glioblastoma multiforme (GBM, Grade IV), have the tendency of diffusely infiltrating the normal brain parenchyma. GBM is characterized by uncontrolled proliferation and resistance to apoptosis, rampant invasion, recalcitrance to most established therapies etc which makes them the most aggressive of all gliomas with a median survival of about 12 months. This makes it imperative to initiate further studies to understand the molecular basis of this disease. Gene expression profiling studies have been central to this effort. In recent years, several Microarray studies have provided crucial insights into the biological role of novel genes not previously associated with astrocytomas. In a previous Microarray study, several differentially regulated genes in astrocytoma were identified in our laboratory. In addition to many up regulated genes, several down regulated genes were also identified in this study. Down regulated genes are interesting to study because of their relevance as possible tumor suppressor genes. Hence, we decided to characterize the regulation and functional significance of few down regulated genes. The specific objectives of the study are as follows 1)To validate novel down‐regulated genes in astrocytomas identified by a previous Microarray study. 2)To understand the mechanism of down-regulation of a few selected gene. 3)Functional characterization of DIRAS2, a novel astrocytoma down‐regulated gene with respect to its possible role in astrocytoma progression. Towards these objectives, we identified 21 genes as differentially down-regulated across all grades of astrocytoma based on a previous Microarray study from our lab and data from literature. Real time qRT-PCR analysis performed on these 21 genes confirmed their down-regulation in all grades of astrocytoma as compared to normal brain tissues. From these 21 genes, we short-listed 10 of the most consistently down-regulated genes for further analysis. These genes were DIRAS2, IGFBP9, MAL2, MBP, OLFM1, PACSIN1, RAB26, SYT1, SYT5 and VSNL1. We also confirmed the expression of two of the genes, OLFM1 and RAB26 at the protein level by performing immunohistochemical analysis on an independent set of 38 tissues that included 10 normal tissues and 28 tissues from different grades of astrocytoma. OLFM1 was found to be down-regulated in a grade specific manner. RAB26 expression was found to be strikingly high in all the low grade astrocytomas in comparison to high grade astrocytomas which made it an interesting gene to study functionally. On functional characterization, we found that RAB26 over‐expressing LN229 cells showed significantly reduced invasion compared to the vector transfected cells suggesting RAB26 could have a tumor-suppressing role in astrocytomas. In order to investigate whether transcriptional modulation could play a role in the down-regulation of these 10 genes, we searched for transcription factor binding sites in approximately 2kb 5’ flanking region of each gene. Intriguingly one or more PAX6 binding sites were present in all their promoters. In light of the fact that PAX6 has been proposed as a tumor‐suppressor in astrocytomas, we predicted that some of these genes could be targets of PAX6 transactivation and could possibly mediate some of the tumor‐suppressive actions of PAX6. PAX6 has been proposed as a down stream target of Notch signaling in the context of eye development. Similar to this observation, upon activation of Notch signaling with a virus expressing human intracellular domain of Notch (Ad-NIC-1), PAX6 expression was found to be induced in glioma cell lines suggesting PAX6 to be a novel NOTCH target in astrocytomas. In addition, Ad-NIC-1 infection could also induce the expression of OLFM1, RAB26, MAL2 and MBP in U343 cells. We could also demonstrate that Ad-NIC-1 co-operates with PAX6 in the regulation of these four genes in cell lines expressing endogenous PAX6, namely U343 and U251. Intriguingly, in a cell-line lacking PAX6 expression (LN229), Ad-NIC-1 could not induce OLFM1, RAB26 and MBP, although we could see induction of MAL2. Interestingly, PAX6 overexpression in LN229 cells in the absence of Ad-NIC-1 could induce OLFM1, RAB26 and MAL2. In contrast, infection of Ad-NIC-1 on the PAX6 over-expressing cells seemed to have an antagonistic effect on the expression of OLFM1, RAB26 and MBP, suggesting that Ad-NIC-1 antagonizes PAX6 actions in these cells. Ad-NIC-1 infection resulted in increased apoptosis in a PAX6 independent manner in U343 cells, which as previously mentioned has high levels of PAX6 endogenous expression. Conversely, Ad-NIC-1 could not induce apoptosis in LN229 cells, which has negligible expression of PAX6. We could also demonstrate that apoptosis induced in U343 cells could be in a p53 dependent manner. Activation of AMPK pathway and inhibition of the mTOR pathway as a consequence of p53 induction could also explain the Ad-NIC-1 mediated apoptosis that was seen in these cells. Thus, we have proposed that Notch signaling could possibly have a tumor-suppressing role in the presence of PAX6. We also suggest that down-regulation of OLFM1, RAB26, MAL2 and MBP via the NOTCH-PAX6 axis could be a possible molecular mechanism for the down-regulation of these genes. With respect to the third objective, we sought to characterize DIRAS2 with respect to its function in astrocytomas. DIRAS2 was identified as a down‐regulated gene in all grades of astrocytoma by our Microarray study. We were also able to validate the down‐regulation of DIRAS2 in all grades of astrocytomas. DIRAS2 also bears significant homology to RIG1 (also known as DIRAS1), which has been proposed as a tumor suppressor gene in astrocytomas. In the light of these data, we predicted that DIRAS2 could be a tumor suppressor gene in astrocytomas. Overexpression of DIRAS2 in two glioma cell lines U87 and C6 did not reveal any appreciable change in proliferation. Strikingly when the DIRAS2 over-expressing clones were grown in the absence of serum, there was marked increase in proliferation with respect to vector transfected clones along with a distinct change in morphology. Decorin expression in the DIRAS2 over-expressing clones was found to be up regulated and could be responsible for the altered morphology as well as enhanced viability in absence of serum. Interestingly along with Decorin expression, we also observed an increase in phosphor-SMAD2 levels indicative of activated TGF‐β signaling in the DIRAS2 over-expressing clones in the absence of serum. In the soft agar and migration/invasion assays, the results across the two cell lines, U87 and C6 were contrasting. DIRAS2 over-expressing clones of U87 cells formed visibly larger and increased number of colonies as compared to vector transfected clones and there was about a three fold increase in invasion with respect to that seen in vector transfected clones in the matrigel invasion assay. On the other hand, DIRAS2 over-expressing C6 clones formed colonies of smaller size compared to vector transfected clones and a marked decrease in migration was observed in the DIRAS2 over-expressing clones of C6. The discrepancies in the results in these two cell lines could be attributed to the presence of other regulators of DIRAS2 function unique to each of the two cell lines. Although in the present study, the results with respect to its predicted function as a tumor-suppressor gene has not been conclusive, the role of DIRAS2 in tumorigenesis may depend on the cellular context in which the protein is expressed. Overall in this study, we have identified a novel down regulated gene signature in astrocytomas consisting of OLFM1, RAB26, MAL2 and MBP. Furthermore, we have proposed that inhibition of NOTCH and PAX6 signaling pathways could be responsible for the down-regulated expression of OLFM1, RAB26, MAL2 and MBP in astrocytomas. Collectively, these results suggest that astrocytomas with activated Notch1 and/or Pax6 signaling could have good prognosis due to the tumor suppressive actions of OLFM1, RAB26, MAL2 and MBP

Comme décrit dans la partie But du Travail, les tumeurs gliales sont particulièrement agressives d’un point de vue clinique. Le glioblastome, qui correspond au grade de malignité le plus élevé des gliomes, est associé à un pronostic très sombre car aucun patient atteint de ce cancer n’a pu être guéri à ce jour. Ces tumeurs envahissent de manière diffuse (par essaimage de cellules tumorales isolées) le parenchyme cérébral, ce qui empêche une résection chirurgicale complète de la tumeur. De plus, les cellules tumorales gliales d’origine astrocytaire sont souvent résistantes à l’apoptose et donc aux thérapies adjuvantes telles que la chimiothérapie et la radiothérapie. La galectine-1 est une petite protéine intervenant directement dans les processus migratoires des cellules gliales tumorales. Nous avons donc poursuivi la caractérisation des rôles biologiques que pourrait exercer la galectine-1 au sein des gliomes.<p>Nous avons tout d’abord montré que la galectine-1 est impliquée dans la chimiorésistance des gliomes. En effet, nous avons démontré que la diminution du taux d’expression de la galectine-1, au moyen d’un siRNA au sein d’un modèle de gliome expérimental, permet d’augmenter le bénéfice thérapeutique du témozolomide in vivo sans toutefois induire d’apoptose, d’autophagie ou de perméabilisation de la membrane des lysosomes. Nous avons également montré que la diminution du taux d’expression de la galectine-1 au sein de ce modèle de gliome expérimental affecte les processus d’angiogenèse in vivo et de « vasculogenic mimicry » in vitro. Nous avons identifié la protéine ORP150 comme l’une des principales cibles de l’effet pro-angiogénique de la galectine-1, sachant que la protéine ORP150 contrôle la maturation du facteur VEGF. Nous avons ensuite montré que le rôle de la galectine-1 dans la chimiorésistance des gliomes et dans l’angiogenèse est directement lié à l’implication de la galectine-1 dans le processus de réponse au stress du réticulum endoplasmique. Via ce processus, la galectine-1 modulerait l’expression d’un certain nombre de gènes tels que ATF3, DUSP5 et HERP, qui sont impliqués dans la chimiorésistance et des gènes tels que ORP150 et MDG1 qui sont impliqués dans l’angiogenèse. <p>Enfin, nous avons également montré que la galectine-1 régule l’expression du gène BEX2 et que celui-ci joue un rôle important dans la biologie des gliomes, notamment dans les processus d’angiogenèse et de migration cellulaire. <p>En conclusion, notre travail suggère que l’étiquette « biomarqueur » pourrait être attribuée à la galectine-1 pour qualifier l’agressivité biologique des gliomes malins et que la galectine-1 pourrait représenter une nouvelle cible thérapeutique dans le combat contre les gliomes malins en général, et le glioblastome en particulier.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Multi-omics high-throughput technologies produce data sets which are not restricted to only one but consist of multiple omics modalities, often as patient-matched tumour specimens. The integrative analysis of these omics modalities is essential to obtain a holistic view on the otherwise fragmented information hidden in this data. We present an intuitive method enabling the combined analysis of multi-omics data based on self-organizing maps machine learning. It “portrays” the expression, methylation and copy number variations (CNV) landscapes of each tumour using the same gene-centred coordinate system. It enables the visual evaluation and direct comparison of the different omics layers on a personalized basis. We applied this combined molecular portrayal to lower grade gliomas, a heterogeneous brain tumour entity. It classifies into a series of molecular subtypes defined by genetic key lesions, which associate with large-scale effects on DNA methylation and gene expression, and in final consequence, drive with cell fate decisions towards oligodendroglioma-, astrocytoma- and glioblastoma-like cancer cell lineages with different prognoses. Consensus modes of concerted changes of expression, methylation and CNV are governed by the degree of co-regulation within and between the omics layers. The method is not restricted to the triple-omics data used here. The similarity landscapes reflect partly independent effects of genetic lesions and DNA methylation with consequences for cancer hallmark characteristics such as proliferation, inflammation and blocked differentiation in a subtype specific fashion. It can be extended to integrate other omics features such as genetic mutation, protein expression data as well as extracting prognostic markers.

Molecular mechanisms of lower-grade (II–III) diffuse gliomas (LGG) are still poorly understood, mainly because of their heterogeneity. They split into astrocytoma- (IDH-A) and oligodendroglioma-like (IDH-O) tumors both carrying mutations(s) at the isocitrate dehydrogenase (IDH) gene and into IDH wild type (IDH-wt) gliomas of glioblastoma resemblance. We generated detailed maps of the transcriptomes and DNA methylomes, revealing that cell functions divided into three major archetypic hallmarks: (i) increased proliferation in IDH-wt and, to a lesser degree, IDH-O; (ii) increased inflammation in IDH-A and IDH-wt; and (iii) the loss of synaptic transmission in all subtypes. Immunogenic properties of IDH-A are diverse, partly resembling signatures observed in grade IV mesenchymal glioblastomas or in grade I pilocytic astrocytomas. We analyzed details of coregulation between gene expression and DNA methylation and of the immunogenic micro-environment presumably driving tumor development and treatment resistance. Our transcriptome and methylome maps support personalized, case-by-case views to decipher the heterogeneity of glioma states in terms of data portraits. Thereby, molecular cartography provides a graphical coordinate system that links gene-level information with glioma subtypes, their phenotypes, and clinical context.

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.

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