ASSESSMENT OF INTRACELLULAR REDOX-BALANCE ROLE IN TMZ-RESISTANCE-RELATED CYTOPROTECTIVE PATHWAYS IN GBM

Background: Glioblastoma (GBM) represents the most aggressive astrocytic brain tumour in adults and exhibits a dismal prognosis related to resistance to therapy, which is principally based on Temozolomide plus RT (Stupp protocol). Among crucial factors which are involved in resistance to TMZ treatment, HIF-1\u3b1 emerges since it activates several pathways, among which angiogenesis and EMT. Beyond its primary alkylating power, TMZ can exerts other secondary effects within cells, as ER stress-mediated ROS release from mitochondria and activation of autophagy. Several works have described HIF-1\u3b1 degradation in lysosomes through the chaperone mediated autophagy (CMA) pathway. Importance of this indirect therapeutic potential has to be yet fully elucidated but the comprehension of the main mechanisms is shedding new light on tumour resistance strategies. At the same time, new procedures for the improvement of radiotherapy treatments could help the development of more precise approaches. Aims: The main purposes of this research project have been: to understand the molecular mechanisms underlying GBM resistance to TMZ, to investigate HIF-1\u3b1 role in resistance and to assess a new strategy able to restore sensitivity to this treatment. Moreover, since the need for a murine model of resistant GBM to test the efficacy of the new therapeutic strategy, a secondary aim of this project has been the setting up of an orthotopic model of GBM, resistant to TMZ, and to characterize it as regards its radio-responsiveness. Final aim of the study has been to use the same orthotopic model to set up a non-invasive procedure to assess GBM by means of PET using 18F-Fluciclovine, whose cellular uptake is dependent upon the rate of activity of amino acid transporters (ASCT2). Materials and methods: HIF-1\u3b1 activity crucial role in responsiveness to TMZ has been evaluated characterising two human GBM cell lines, U251-responsive cells to TMZ and T98-resistant ones, through molecular, biochemical ang gene expression analyses, by means of gene silencing and PX-478-mediated pharmacological inhibition. Moreover, CMA engagement in responsiveness to TMZ has been evaluated, following the experimental approach used for HIF-1\u3b1 and through biochemical and protein studies of CMA pathway. Mitochondrial ROS contribution and the detox machinery role in TMZ treatment have been investigated, respectively, treating cells with MitoT utilising all the aforementioned techniques (including scratch test) and by means of gene expression profile and protein analyses. H2O2 treatments, which retrace the experimental approach used for MitoT, have been used for testing the potential ROS role in reverting resistance to TMZ. Murine glioma cell line CT-2A, after having been characterised as U251 and T98 for evaluating its responsiveness to TMZ, have been stereotaxically injected (i.c.) in C57BL/6J mice to set up orthotopic glioma models. MRI scans have been carried out at days 9 and 15 after i.c. injection for monitoring tumour growth. MRI has been exploited also for monitoring tumour growth after RT (15Gy-hemibrain). 18F-Fluciclovine-PET has been performed 16 days after i.c. injection. Untreated and RT-treated mice brains have been collected for IHC analyses regarding ASCT2 expression levels. Results: This study allowed the identification of response biomarkers and mechanisms involved in GBM resistance to TMZ treatment. HIF-1\u3b1 has been identified as a crucial factor in resistance to TMZ: hypoxic cells are characterized by a lower sensitivity to the drug, while a significant decrease both in viability and HIF-1\u3b1 activity has been detected after treatment in sensitive cells while no modulation in HIF-1\u3b1 activity and viability was observed in T98 resistant cells. These results were confirmed by assessing also apoptosis-, CMA- and EMT-related gene expression. Further results showed the involvement of CMA in HIF-1\u3b1 degradation and the consequent cytotoxicity due to the drug: in fact, LAMP-2A silencing induced resistance in previously sensitive cells, while HIF-1\u3b1 gene silencing reverted T98 phenotype from a previously resistant to a sensitive one. Also, the PX-478 mediated HIF-1\u3b1 activity abrogation confirmed the previous result. CMA activation following TMZ treatment can be induced by ROS release from the mitochondria as demonstrated by using the MitoT. The study of detox machinery showed a down-regulation in sensitive cells after TMZ treatment, consistently with the temporary ROS fluctuations. At the same time, an external-mediated increase in intracellular ROS level by H2O2 treatment resulted to be able to induce the same mechanisms activated by TMZ in sensitive cells after ROS release, determining a responsive profile both in sensitive and resistant cells. Of note, here we report that, only the concurrent treatment with H2O2 and TMZ induced a completely significant responsive profile in resistant cells, confirming the crucial role of ROS, CMA, HIF-1\u3b1 modulation and the drug. The final result of this work is the development of a TMZ-resistant murine orthotopic model by i.c. injection of CT-2A cells. This model resulted to be responsive to RT, as reported by MRI studies. 18F-Fluciclovine tracer was used to monitor tumour extent in view of its implementation on the evaluation of response to treatment by PET. Finally, preliminary IHC data of untreated and RT-treated mice brains have confirmed a down-regulation of ASCT2 expression after RT compared to the untreated ones. Conclusions: In this thesis work, it has been demonstrated that HIF-1\u3b1 activity is a key player in resistance to TMZ, that CMA has a crucial role in mediating TMZ-induced cytotoxicity and that the induction of this pathway was due to a transitory increase in intracellular ROS level. Moreover, an exogenous increase in ROS levels has been able to restore a completely responsive profile in resistant cells opening the way for the evaluation of new therapeutic approaches. CT-2A orthotopic murine models represent a good opportunity for the in vivo assessment of these new treatments, especially using the non-invasive multimodal imaging, strategy described herein