Introduction: Glioblastoma (GBM), the most aggressive form of malignant gliomas, is an incurable tumor of the central nervous system which starts in glial cells, and rapidly diffuses through the brain. Surgical resection, radiotherapy and chemotherapy are the standard of care of GBM, improving the survival rate up to only 14-15 months. The high infiltrating nature of the tumor hampers surgical resection and tumoral cells often develop chemoresistance to the widely-used temozolomide (TMZ). Currently, a targeted therapy is not available. Therefore, the 5 years survival rate of these patients is extremely poor and does not exceed 5%. Most GBMs result from constitutive activation of the epidermal growth factor receptor (EGFR) and mutation in the pro-oncogenic phosphoinositide 3-kinase (PI3K) pathways, which are known regulators of cell growth. These pathways are extremely conserved in fruit fly: Because of this, we used Drosophila melanogaster as a model system to recapitulate in vivo certain tumoral features of GBMs. As the human one, Drosophila glia is constituted of several types of glial cells, which are involved in the formation of blood-brain-barrier and in phagocytosis of neural debris. In Drosophila glial precursor, we over-express a mutated form of the human EGFR and of Dp110, a regulator of PI3K signaling. Such manipulations result in massive hyperplasticity of the larval brain, due to an expansion of the glial compartment. Autophagy is a conserved catabolic process that provides nutrients by degradation of cellular constituents in lysosomes. How autophagy acts in tumor growth is currently unclear and the focus of intense investigation. Aim: We used a Drosophila glioma model to understand in vivo the role of the autophagy-lysosomal pathway in tumor growth and to identify new potential therapeutic targets suitable for clinical translation. Materials and methods: Drosophila 3rd instar larval brains were used to assess tumor growth and to monitor the autophagy-lysosomal pathway. Larvae were grown on standard fly food until the 3rd instar stage and larval brains were isolated and processed for the subsequent experiments. RNA was extracted by Mini-RNeasy kit from Qiagen, while proteins were extracted using RIPA buffer. Disaggregation of larval brains was carried out using Rinaldini’s solution and proteases. For immunofluorescence acquisitions, brains were isolated from larvae and processed with specific antibodies. Down-regulation of a targeted gene was performed using RNAi fly lines. Results: The excessive glial growth affects non-cell autonomously the neural tissue, which appears reduced and morphologically altered. Moreover, tumor growth impairs larval behavior and leads to death. Interestingly, we found that compared to control, the hyperplastic tissue displays a transcriptional and protein up-regulation of the autophagy adapter ref(2)P, the Drosophila p62/SQSTM1. However, canonical regulators of autophagy, controlled by Mitf, the Drosophila TFEB, are not deregulated in the glial tumor, compared to control. This suggests that gliomas might not have the ability to induce the autophagy-lysosomal pathway to supply to their growth needs. Moreover, we observed that the lysosomal compartment is expanded but functional. In contrast, we found that down-regulation of a component of the vacuolar-H+ATPase (V-ATPase), which is required for lysosomal acidification, reduces tumor growth. Surprisingly, ref(2) protein and transcriptional levels are restored back to physiology in tumoral brain in which the component is down-regulated. Importantly, we found that depletion prevents most of glia overgrowth and decreases activity of the PI3K pathway. Conclusion: Collectively, our data indicate that autophagy might be limiting for tumor growth and that components of the V-ATPase proton pump might be novel targets for treatment of GBM.

V-ATPASE AND AUTOPHAGY PREVENT GLIOMA GROWTH IN A DROSOPHILA MODEL SYSTEM / M. Formica ; tutore: R. Ghidoni ; co-tutore: T. Vaccari ; coordinatore: R. Ghidoni. - : . DIPARTIMENTO DI SCIENZE DELLA SALUTE, 2019 Jan 15. ((31. ciclo, Anno Accademico 2018. [10.13130/formica-miriam_phd2019-01-15].

V-ATPASE AND AUTOPHAGY PREVENT GLIOMA GROWTH IN A DROSOPHILA MODEL SYSTEM

FORMICA, MIRIAM
2019-01-15

Abstract

Introduction: Glioblastoma (GBM), the most aggressive form of malignant gliomas, is an incurable tumor of the central nervous system which starts in glial cells, and rapidly diffuses through the brain. Surgical resection, radiotherapy and chemotherapy are the standard of care of GBM, improving the survival rate up to only 14-15 months. The high infiltrating nature of the tumor hampers surgical resection and tumoral cells often develop chemoresistance to the widely-used temozolomide (TMZ). Currently, a targeted therapy is not available. Therefore, the 5 years survival rate of these patients is extremely poor and does not exceed 5%. Most GBMs result from constitutive activation of the epidermal growth factor receptor (EGFR) and mutation in the pro-oncogenic phosphoinositide 3-kinase (PI3K) pathways, which are known regulators of cell growth. These pathways are extremely conserved in fruit fly: Because of this, we used Drosophila melanogaster as a model system to recapitulate in vivo certain tumoral features of GBMs. As the human one, Drosophila glia is constituted of several types of glial cells, which are involved in the formation of blood-brain-barrier and in phagocytosis of neural debris. In Drosophila glial precursor, we over-express a mutated form of the human EGFR and of Dp110, a regulator of PI3K signaling. Such manipulations result in massive hyperplasticity of the larval brain, due to an expansion of the glial compartment. Autophagy is a conserved catabolic process that provides nutrients by degradation of cellular constituents in lysosomes. How autophagy acts in tumor growth is currently unclear and the focus of intense investigation. Aim: We used a Drosophila glioma model to understand in vivo the role of the autophagy-lysosomal pathway in tumor growth and to identify new potential therapeutic targets suitable for clinical translation. Materials and methods: Drosophila 3rd instar larval brains were used to assess tumor growth and to monitor the autophagy-lysosomal pathway. Larvae were grown on standard fly food until the 3rd instar stage and larval brains were isolated and processed for the subsequent experiments. RNA was extracted by Mini-RNeasy kit from Qiagen, while proteins were extracted using RIPA buffer. Disaggregation of larval brains was carried out using Rinaldini’s solution and proteases. For immunofluorescence acquisitions, brains were isolated from larvae and processed with specific antibodies. Down-regulation of a targeted gene was performed using RNAi fly lines. Results: The excessive glial growth affects non-cell autonomously the neural tissue, which appears reduced and morphologically altered. Moreover, tumor growth impairs larval behavior and leads to death. Interestingly, we found that compared to control, the hyperplastic tissue displays a transcriptional and protein up-regulation of the autophagy adapter ref(2)P, the Drosophila p62/SQSTM1. However, canonical regulators of autophagy, controlled by Mitf, the Drosophila TFEB, are not deregulated in the glial tumor, compared to control. This suggests that gliomas might not have the ability to induce the autophagy-lysosomal pathway to supply to their growth needs. Moreover, we observed that the lysosomal compartment is expanded but functional. In contrast, we found that down-regulation of a component of the vacuolar-H+ATPase (V-ATPase), which is required for lysosomal acidification, reduces tumor growth. Surprisingly, ref(2) protein and transcriptional levels are restored back to physiology in tumoral brain in which the component is down-regulated. Importantly, we found that depletion prevents most of glia overgrowth and decreases activity of the PI3K pathway. Conclusion: Collectively, our data indicate that autophagy might be limiting for tumor growth and that components of the V-ATPase proton pump might be novel targets for treatment of GBM.
GHIDONI, RICCARDO
GHIDONI, RICCARDO
Drosophila melanogaster ; V-ATPase ; Glioblastoma ; Autophagy ; PI3K ; EGFR
Settore BIO/11 - Biologia Molecolare
V-ATPASE AND AUTOPHAGY PREVENT GLIOMA GROWTH IN A DROSOPHILA MODEL SYSTEM / M. Formica ; tutore: R. Ghidoni ; co-tutore: T. Vaccari ; coordinatore: R. Ghidoni. - : . DIPARTIMENTO DI SCIENZE DELLA SALUTE, 2019 Jan 15. ((31. ciclo, Anno Accademico 2018. [10.13130/formica-miriam_phd2019-01-15].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/611790
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