Glioblastoma is the most common primary malignant brain tumour in the adult population. Despite multimodality treatment with surgery, radiotherapy and chemotherapy, outcomes are very poor, with less than 15% of patients alive after two years. Increasing evidence suggests that Glioma stem cells (GSCs) are likely to play an important role in the biology of this disease and are involved in treatment resistance and tumour recurrence following standard therapy. GSCs are characterized by enhanced self-renewal, highlighted by the expression of stem cell markers, such as CD133 and Nestin, elevated invasive behaviour, chemo and radiotherapy resistance, and the ability to generate multi-lineage progenities. A typical feature of GSCs is also the elevated chromosomal instability (CIN): they are characterized by various numerical and structural aberrations, deletions, amplification and loss of heterozygosity. A variety of alterations have been proposed as being responsible for CIN, including defects in genes involved in the regulation of the mitotic machinery, such as the Aurora Kinases, making them a promising therapeutic target for GSCs depletion. My thesis address two main aspects of this research area, aiming at the identification of new GSCs-targeted therapeutic strategies for GBM complete eradication. In the first part of my project I investigated the effect of Danusertib, a pan-Aurora kinases inhibitor on 5 GSC lines isolated from glioblastoma patients, previously characterized in our laboratory from a cytogenomic and epigenomic point of view. Results showed that response to Danusertib exposure was heterogeneous among GSC lines. Some of them were more sensitive to subtle changes in Aurora kinases activity, which result in huge morphological alterations, a rapid increase in polyploidy and subsequently in senescence, with a consistent reduction in clonogenic survival and proliferation. Interestingly I also observed that the more resistant cell lines showed an increase in ploidy and senescence after repeated rounds of Danusertib exposure, suggesting that there could be the presence of an intolerable ploidy threshold that leads cells to senescence. In the second part of my thesis I presented some preliminary results I achieved in Dr Hochegger’s lab (Genome Damage and Stability Center, University of Sussex, Brighton, UK), where I took part in a project aimed on setting up CrispR/Cas9 mediated GFP or RFP-tagged CD133 (PROM1 gene) and Nestin (NES gene) glioma stem cell lines in order to look, with live cell imaging techniques, for signs for asymmetric cell division, by which a single GSC would be able to both maintain a pool of self-renewing stem cells and produce differential progeny, using live cell imaging. The biological significance of asymmetric or symmetric division modes is not yet fully understood, but improved understanding of this phenomenon may lead to the development of preventative treatments or improved therapeutic options for brain tumour patients through the identification of novel targets that are involved in the control of asymmetric cell division in human brain tissue.

Il glioblastoma multiforme rappresenta la forma più frequente e maligna fra i tumori cerebrali primari. Nonostante un approccio terapeutico multimodale, che include resezione chirurgica, radio e chemioterapia, la prognosi è generalmente infausta. La maggior parte dei pazienti muore dopo soli 12-15 mesi dalla diagnosi e la percentuale di recidiva è pari all’80%. Crescenti evidenze suggeriscono che una delle principali cause della comparsa di recidive e del fallimento delle attuali strategie terapeutiche è la presenza, all’interno del tumore, di una sottopopolazione di cellule tumorali con caratteristiche staminali, chiamate glioma stem cells (GSCs). Le GSCs sono caratterizzate da un aumentato self-renewal, come dimostrato dall’espressione di tipici marcatori di staminalità, quali CD133 e Nestina, da un’elevata invasività, dalla capacità di differenziare in diversi lineages neurali e da una spiccata chemo e radio resistenza. Una tipica caratteristica delle GSCs è anche l’elevata instabilità cromosomica (CIN). Infatti, tali cellule, presentano numerose alterazioni numeriche e strutturali, delezioni, amplificazioni e perdite di eterozigosità. Diverse alterazioni sono state indicate come responsabili dell’instabilità cromosomica, tra cui difetti in geni codificanti per proteine coinvolte nel macchinario mitotico, come le Aurora chinasi, rendendole un possibile e promettente target terapeutico. La mia tesi si propone, perciò, di indagare due aspetti principali di quest’area di ricerca, con l’obiettivo di individuare nuove possibili strategie terapeutiche GSC-targeted, necessarie per una completa eradicazione del GBM. Nella prima parte del mio progetto ho indagato l’effetto di Danusertib, un inibitore delle Aurora chinasi, su 5 linee di cellule staminali tumorali isolate da glioblastoma, precedentemente caratterizzate nel nostro laboratorio da un punto di vista citogenomico ed epigenomico. I risultati ottenuti hanno mostrato una risposta eterogenea delle diverse linee cellulari all’inibitore: alcune di esse sono risultate maggiormente sensibili, mostrando evidenti alterazioni della morfologia, un considerevole aumento della ploidia e la presenza di un fenotipo senescente associato ad una riduzione del potenziale clonogenico e della proliferazione cellulare. Tuttavia un risultato particolarmente interessante è che anche le linee cellulari più resistenti dopo diversi rounds di esposizione all’inibitore diventano maggiormente sensibili, suggerendo la presenza di una sorta di soglia della plodia superata la quale le cellule vengono indotte alla senescenza. Nella seconda parte della mia tesi ho presentato alcuni dati preliminari ottenuti nel laboratorio del Dr. Hochegger (Genome Damage and Stability Center, University of Sussex, Brighton, UK), dove ho trascorso sei mesi durante il dottorato. Durante questo periodo ho preso parte ad un progetto mirato a sviluppare alcune linee di GSCs in cui i marcatori di staminalità CD133 e Nestina sono taggati con proteine fluorescenti attraverso la tecnica CrispR/Cas9. L’obiettivo è quello di utilizzare tali linee cellulari modificate geneticamente per studiare attraverso tecniche di live imaging la modalità di divisione cellulare messa in atto dalle GSCs, attraverso la quale esse sarebbero in grado, non solo di mantenere in pool di cellule staminali tumorali, ma anche di generare cellule più differenziate che costituiscono la maggior parte della massa tumorale. La comprensione di tale modalità di divisione cellulare potrebbe favorire l’individuazione di nuove strategie terapeutiche, attraverso l’individuazione di nuovi target coinvolti nel controllo della divisione asimmetrica delle GSCs nel tessuto cerebrale.

(2017). CHROMOSOMAL INSTABILITY IN GLIOMA STEM CELL LINES FROM GLIOBLASTOMA MULTIFORME: IMPLICATIONS FOR NEW THERAPEUTIC STRATEGIES. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).

CHROMOSOMAL INSTABILITY IN GLIOMA STEM CELL LINES FROM GLIOBLASTOMA MULTIFORME: IMPLICATIONS FOR NEW THERAPEUTIC STRATEGIES

CILIBRASI, CHIARA
2017

Abstract

Glioblastoma is the most common primary malignant brain tumour in the adult population. Despite multimodality treatment with surgery, radiotherapy and chemotherapy, outcomes are very poor, with less than 15% of patients alive after two years. Increasing evidence suggests that Glioma stem cells (GSCs) are likely to play an important role in the biology of this disease and are involved in treatment resistance and tumour recurrence following standard therapy. GSCs are characterized by enhanced self-renewal, highlighted by the expression of stem cell markers, such as CD133 and Nestin, elevated invasive behaviour, chemo and radiotherapy resistance, and the ability to generate multi-lineage progenities. A typical feature of GSCs is also the elevated chromosomal instability (CIN): they are characterized by various numerical and structural aberrations, deletions, amplification and loss of heterozygosity. A variety of alterations have been proposed as being responsible for CIN, including defects in genes involved in the regulation of the mitotic machinery, such as the Aurora Kinases, making them a promising therapeutic target for GSCs depletion. My thesis address two main aspects of this research area, aiming at the identification of new GSCs-targeted therapeutic strategies for GBM complete eradication. In the first part of my project I investigated the effect of Danusertib, a pan-Aurora kinases inhibitor on 5 GSC lines isolated from glioblastoma patients, previously characterized in our laboratory from a cytogenomic and epigenomic point of view. Results showed that response to Danusertib exposure was heterogeneous among GSC lines. Some of them were more sensitive to subtle changes in Aurora kinases activity, which result in huge morphological alterations, a rapid increase in polyploidy and subsequently in senescence, with a consistent reduction in clonogenic survival and proliferation. Interestingly I also observed that the more resistant cell lines showed an increase in ploidy and senescence after repeated rounds of Danusertib exposure, suggesting that there could be the presence of an intolerable ploidy threshold that leads cells to senescence. In the second part of my thesis I presented some preliminary results I achieved in Dr Hochegger’s lab (Genome Damage and Stability Center, University of Sussex, Brighton, UK), where I took part in a project aimed on setting up CrispR/Cas9 mediated GFP or RFP-tagged CD133 (PROM1 gene) and Nestin (NES gene) glioma stem cell lines in order to look, with live cell imaging techniques, for signs for asymmetric cell division, by which a single GSC would be able to both maintain a pool of self-renewing stem cells and produce differential progeny, using live cell imaging. The biological significance of asymmetric or symmetric division modes is not yet fully understood, but improved understanding of this phenomenon may lead to the development of preventative treatments or improved therapeutic options for brain tumour patients through the identification of novel targets that are involved in the control of asymmetric cell division in human brain tissue.
BENTIVEGNA, ANGELA
Glioblastoma,; chromosomal; instability,; Aurora; kinases
Glioblastoma,; chromosomal; instability,; Aurora; kinases
BIO/16 - ANATOMIA UMANA
English
3-apr-2017
NEUROSCIENZE - 90R
29
2015/2016
open
(2017). CHROMOSOMAL INSTABILITY IN GLIOMA STEM CELL LINES FROM GLIOBLASTOMA MULTIFORME: IMPLICATIONS FOR NEW THERAPEUTIC STRATEGIES. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/158150
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