AMPK activation protects astrocytes from hypoxia­induced cell death.

Adenosine monophosphate (AMP)­activated protein kinase (AMPK) is a major cellular energy sensor that is activated by an increase in the AMP/adenosine triphosphate (ATP) ratio. This causes the initiation of adaptive cellular programs, leading to the inhibition of anabolic pathways and increasing ATP synthesis. AMPK indirectly inhibits mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a serine/threonine kinase and central regulator of cell growth and metabolism, which integrates various growth inhibitory signals, such as the depletion of glucose, amino acids, ATP and oxygen. While neuroprotective approaches by definition focus on neurons, that are more sensitive under cell stress conditions, astrocytes play an important role in the cerebral energy homeostasis during ischemia. Therefore, the protection of astrocytic cells or other glial cells may contribute to the preservation of neuronal integrity and function. In the present study, it was thus hypothesized that a preventive induction of energy deprivation­activated signaling pathways via AMPK may protect astrocytes from hypoxia and glucose deprivation. Hypoxia­induced cell death was measured in a paradigm of hypoxia and partial glucose deprivation in vitro in the immortalized human astrocytic cell line SVG. Both the glycolysis inhibitor 2­deoxy­d­glucose (2DG) and the AMPK activator A­769662 induced the phosphorylation of AMPK, resulting in mTORC1 inhibition, as evidenced by a decrease in the phosphorylation of the target ribosomal protein S6 (RPS6). Treatment with both 2DG and A­769662 also decreased glucose consumption and lactate production. Furthermore, A­769662, but not 2DG induced an increase in oxygen consumption, possibly indicating a more efficient glucose utilization through oxidative phosphorylation. Hypoxia­induced cell death was profoundly reduced by treatment with 2DG or A­769662. On the whole, the findings of the present study demonstrate, that AMPK activation via 2DG or A­769662 protects astrocytes under hypoxic and glucose­depleted conditions.

Disruption of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α reverts key features of the neoplastic phenotype of glioma cells.

The peroxisome proliferator-activated receptor γ coactivator (PGC)-1α is a master regulator of mitochondrial biogenesis and controls metabolism by coordinating transcriptional events. Here, we interrogated whether PGC-1α is involved in tumor growth and the metabolic flexibility of glioblastoma cells. PGC-1α was expressed in a subset of established glioma cell lines and primary glioblastoma cell cultures. Furthermore, a higher PGC-1α expression was associated with an adverse outcome in the TCGA glioblastoma dataset. Suppression of PGC-1α expression by shRNA in the PGC-1α-positive U343MG glioblastoma line suppressed mitochondrial gene expression, reduced mitochondrial membrane potential, and diminished oxygen as well as glucose consumption, and lactate production. Compatible with the known PGC-1α functions in reactive oxygen species (ROS) metabolism, glioblastoma cells deficient in PGC-1α displayed ROS accumulation, had reduced RNA levels of proteins involved in ROS detoxification, and were more susceptible to death induction by H2O2 compared with control cells. PGC-1αsh cells also had impaired proliferation and migration rates in vitro and displayed less stem cell characteristics. Complementary effects were observed in PGC-1α-low LNT-229 cells engineered to overexpress PGC-1α. In an in vivo xenograft experiment, tumors formed by U343MG PGC-1αsh glioblastoma cells grew much slower than control tumors and were less invasive. Interestingly, the PGC-1α knockdown conferred protection against hypoxia-induced cell death, probably as a result of less active anabolic pathways, and this effect was associated with reduced epidermal growth factor expression and mammalian target of rapamycin signaling. In summary, PGC-1α modifies the neoplastic phenotype of glioblastoma cells toward more aggressive behavior and therefore makes PGC-1α a potential target for anti-glioblastoma therapies.

Gene Suppression of Transketolase-Like Protein 1 (TKTL1) Sensitizes Glioma Cells to Hypoxia and Ionizing Radiation.

In several tumor entities, transketolase-like protein 1 (TKTL1) has been suggested to promote the nonoxidative part of the pentose phosphate pathway (PPP) and thereby to contribute to a malignant phenotype. However, its role in glioma biology has only been sparsely documented. In the present in vitro study using LNT-229 glioma cells, we analyzed the impact of TKTL1 gene suppression on basic metabolic parameters and on survival following oxygen restriction and ionizing radiation. TKTL1 was induced by hypoxia and by hypoxia-inducible factor-1α (HIF-1α). Knockdown of TKTL1 via shRNA increased the cells' demand for glucose, decreased flux through the PPP and promoted cell death under hypoxic conditions. Following irradiation, suppression of TKTL1 expression resulted in elevated levels of reactive oxygen species (ROS) and reduced clonogenic survival. In summary, our results indicate a role of TKTL1 in the adaptation of tumor cells to oxygen deprivation and in the acquisition of radioresistance. Further studies are necessary to examine whether strategies that antagonize TKTL1 function will be able to restore the sensitivity of glioma cells towards irradiation and antiangiogenic therapies in the more complex in vivo environment.

Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

Glioblastomas are characterized by fast uncontrolled growth leading to hypoxic areas and necrosis. Signalling from EGFR via mammalian target of rapamycin complex 1 (mTORC1) is a major driver of cell growth and proliferation and one of the most commonly altered signalling pathways in glioblastomas. Therefore, epidermal growth factor receptor and mTORC1 signalling are plausible therapeutic targets and clinical trials with inhibitors are in progress. However, we have previously shown that epidermal growth factor receptor and mTORC1 inhibition triggers metabolic changes leading to adverse effects under the conditions of the tumour microenvironment by protecting from hypoxia-induced cell death. We hypothesized that conversely mTORC1 activation sensitizes glioma cells to hypoxia-induced cell death. As a model for mTORC1 activation we used gene suppression of its physiological inhibitor TSC2 (TSC2sh). TSC2sh glioma cells showed increased sensitivity to hypoxia-induced cell death that was accompanied by an earlier ATP depletion and an increase in reactive oxygen species. There was no difference in extracellular glucose consumption but an altered intracellular metabolic profile with an increase of intermediates of the pentose phosphate pathway. Mechanistically, mTORC1 upregulated the first and rate limiting enzyme of the pentose phosphate pathway, G6PD. Furthermore, an increase in oxygen consumption in TSC2sh cells was detected. This appeared to be due to higher transcription rates of genes involved in mitochondrial respiratory function including PPARGC1A and PPARGC1B (also known as PGC-1α and -ß). The finding that mTORC1 activation causes an increase in oxygen consumption and renders malignant glioma cells susceptible to hypoxia and nutrient deprivation could help identify glioblastoma patient cohorts more likely to benefit from hypoxia-inducing therapies such as the VEGFA-targeting antibody bevacizumab in future clinical evaluations.