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Abstract In glioblastoma (GB) cells oxidative stress is induced by both, conditions of the tumor microenvironment as well as by therapeutic interventions. Upregulation of superoxide dismutase 1 (SOD1), a key enzyme for oxidative defense and downstream target of mammalian target of rapamycin complex 1 (mTORC1) is a candidate mechanism to sustain survival and proliferation of tumor cells. SOD1 was inhibited by shRNA mediated gene suppression, CRISPR/Cas9 knockout and pharmacological inhibition in human (primary) GB cells. SOD1 activity was determined by SOD1/2 activity assay. ROS levels, cell death and the NADPH/NADP-ratio were measured under normal and starvation conditions. To study the mTORC1-SOD1 axis, mTORC1 activated TSC2 knockdown cells (TSC2sh) were analyzed. Genetic and pharmacological inhibition of SOD1 correlated with decreased SOD1 activity, increased ROS and enhanced the sensitivity of glioma cells towards starvation- and hypoxia-induced cell death. This was accompanied by a decreased NADPH/NADP-ratio. Furthermore, combination therapy of SOD1 and mTORC1 inhibition partially rescued the protective effect of mTORC1 inhibitor monotherapy. SOD1 mediates adaptation of GB cells to stress conditions in the tumor microenvironment in a mTORC1-dependent manner. Moreover, SOD1 activation contributes to the cell death resistance conferred by mTORC1 inhibitors under hypoxic conditions.

Abstract Glioblastoma is an incurable brain tumor with a median survival below two years. Trials investigating targeted therapy with inhibitors of the kinase mTOR have produced ambiguous results. Especially combination of mTOR inhibition with standard temozolomide radiochemotherapy has resulted in reduced survival in a phase II clinical trial. To date, this phenomenon is only poorly understood. To recreate the therapeutic setting in vitro, we exposed glioblastoma cell lines to co-treatment with rapamycin and temozolomide and assessed cell viability, DNA damage and reactive oxygen species. Additionally, we employed a novel translatomic based mass spectrometry approach (“mePROD”) to analyze acute changes in translated proteins. mTOR inhibition with rapamycin protected glioblastoma cells from temozolomide toxicity. Following co-treatment of temozolomide with rapamycin, an increased translation of reactive oxygen species (ROS)-detoxifying proteins was detected by mass spectrometry. This was accompanied by improved ROS-homeostasis and reduced DNA damage. Additionally, rapamycin induced the expression of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT) in glioblastoma cells with an unmethylated MGMT gene promotor. Inhibition of mTOR antagonized the cytotoxic effects of temozolomide in vitro. The induction of antioxidant defences and MGMT are two underlying candidate mechanisms. Further functional experiments in vitro and in vivo are warranted to characterize this effect that appears relevant for combinatorial therapeutic strategies.

Abstract Although malignant gliomas frequently show aberrant activation of the mammalian target of rapamycin (mTOR), mTOR inhibitors have performed poorly in clinical trials. Besides regulating cell growth and translation, mTOR controls the initiation of autophagy. By recycling cellular components, autophagy can mobilize energy resources, and has thus been attributed cancer-promoting effects. Here, we asked whether the activation of autophagy represents an escape mechanism to pharmacological mTOR inhibition in glioma cells, and explored co-treatment with mTOR and autophagy inhibitors as a therapeutic strategy. Mimicking conditions of the glioma microenvironment, glioma cells were exposed to nutrient starvation and hypoxia. We analyzed autophagic activity, cell growth, viability and oxygen consumption following (co-)treatment with the mTOR inhibitors torin2 or rapamycin, and autophagy inhibitors bafilomycin A1 or MRT68921. Changes in global proteome were quantified by mass spectrometry. In the context of hypoxia and starvation, autophagy was strongly induced in glioma cells and further increased by mTOR inhibition. While torin2 enhanced glioma cell survival, co-treatment with torin2 and bafilomycin A1 failed to promote cell death. Importantly, treatment with bafilomycin A1 alone also protected glioma cells from cell death. Mechanistically, both compounds significantly reduced cell growth and oxygen consumption. Quantitative proteomics analysis showed that bafilomycin A1 induced broad changes in the cellular proteome. More specifically, proteins downregulated by bafilomycin A1 were associated with the mitochondrial respiratory chain and ATP synthesis. Taken together, our results show that activation of autophagy does not account for the cytoprotective effects of mTOR inhibition in our in vitro model of the glioma microenvironment. Our proteomic findings suggest that the pharmacological inhibition of autophagy induces extensive changes in the cellular proteome that can support glioma cell survival under nutrient-deplete and hypoxic conditions. These findings provide a novel perspective on the complex role of autophagy in gliomas.