α-Ketoglutarate supplementation and NAD+ modulation enhance metabolic rewiring and radiosensitization in SLC25A1 inhibited cancer cells.

Metabolic rewiring is the result of the increasing demands and proliferation of cancer cells, leading to changes in the biological activities and responses to treatment of cancer cells. The mitochondrial citrate transport protein SLC25A1 is involved in metabolic reprogramming offering a strategy to induce metabolic bottlenecks relevant to radiosensitization through the accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) upon SLC25A1 inhibition (SLC25A1i). Previous studies have revealed the comparative effects of SLC25A1i or cell-permeable D-2HG (octyl-D-2HG) treatments on DNA damage induction and repair, as well as on energy metabolism and cellular function, which are crucial for the long-term survival of irradiated cells. Here, α-ketoglutarate (αKG), the precursor of D-2HG, potentiated the effects observed upon SLC25A1i on DNA damage repair, cell function and long-term survival in vitro and in vivo, rendering NCI-H460 cancer cells more vulnerable to ionizing radiation. However, αKG treatment alone had little effect on these phenotypes. In addition, supplementation with nicotinamide (NAM), a precursor of NAD (including NAD+ and NADH), counteracted the effects of SLC25A1i or the combination of SLC25A1i with αKG, highlighting a potential importance of the NAD+/NADH balance on cellular activities relevant to the survival of irradiated cancer cells upon SLC25A1i. Furthermore, inhibition of histone lysine demethylases (KDMs), as a major factor affected upon SLC25A1i, by JIB04 treatment alone or in combination with αKG supplementation phenocopied the broad effects on mitochondrial and cellular function induced by SLC25A1i. Taken together, αKG supplementation potentiated the effects on cellular processes observed upon SLC25A1i and increased the cellular demand for NAD to rebalance the cellular state and ensure survival after irradiation. Future studies will elucidate the underlying metabolic reprogramming induced by SLC25A1i and provide novel therapeutic strategies for cancer treatment.

Cigarette smoke causes a bioenergetic crisis in RPE cells involving the downregulation of HIF-1α under normoxia.

Age-related macular degeneration (AMD) is the most common blinding disease in the elderly population. However, there are still many uncertainties regarding the pathophysiology at the molecular level. Currently, impaired energy metabolism in retinal pigment epithelium (RPE) cells is discussed as one major hallmark of early AMD pathophysiology. Hypoxia-inducible factors (HIFs) are important modulators of mitochondrial function. Moreover, smoking is the most important modifiable risk factor for AMD and is known to impair mitochondrial integrity. Therefore, our aim was to establish a cell-based assay that enables us to investigate how smoking affects mitochondrial function in conjunction with HIF signaling in RPE cells. For this purpose, we treated a human RPE cell line with cigarette smoke extract (CSE) under normoxia (21% O2), hypoxia (1% O2), or by co-treatment with Roxadustat, a clinically approved HIF stabilizer. CSE treatment impaired mitochondrial integrity, involving increased mitochondrial reactive oxygen species, disruption of mitochondrial membrane potential, and altered mitochondrial morphology. Treatment effects on cell metabolism were analyzed using a Seahorse Bioanalyzer. Mitochondrial respiration and ATP production were impaired in CSE-treated cells under normoxia. Surprisingly, CSE-treated RPE cells also exhibited decreased glycolytic rate under normoxia, causing a bioenergetic crisis, because two major metabolic pathways that provide ATP were impaired by CSE. Downregulation of glycolytic rate was HIF-dependent because HIF-1α, the α-subunit of HIF-1, was downregulated by CSE on the protein level, especially under normoxia. Moreover, hypoxia incubation and treatment with Roxadustat restored glycolytic flux. Taken together, our in vitro model provides interesting insights into HIF-dependent regulation of glycolysis under normoxic conditions, which will enable us to investigate signaling pathways involved in RPE metabolism in health and disease.

Targeting AKT-Dependent Regulation of Antioxidant Defense Sensitizes AKT-E17K Expressing Cancer Cells to Ionizing Radiation.

Aberrant activation of the phosphatidyl-inositol-3-kinase/protein kinase B (AKT) pathway has clinical relevance to radiation resistance, but the underlying mechanisms are incompletely understood. Protection against reactive oxygen species (ROS) plays an emerging role in the regulation of cell survival upon irradiation. AKT-dependent signaling participates in the regulation of cellular antioxidant defense. Here, we were interested to explore a yet unknown role of aberrant activation of AKT in regulating antioxidant defense in response to IR and associated radiation resistance. We combined genetic and pharmacologic approaches to study how aberrant activation of AKT impacts cell metabolism, antioxidant defense, and radiosensitivity. Therefore, we used TRAMPC1 (TrC1) prostate cancer cells overexpressing the clinically relevant AKT-variant AKT-E17K with increased AKT activity or wildtype AKT (AKT-WT) and analyzed the consequences of direct AKT inhibition (MK2206) and inhibition of AKT-dependent metabolic enzymes on the levels of cellular ROS, antioxidant capacity, metabolic state, short-term and long-term survival without and with irradiation. TrC1 cells expressing the clinically relevant AKT1-E17K variant were characterized by improved antioxidant defense compared to TrC1 AKT-WT cells and this was associated with increased radiation resistance. The underlying mechanisms involved AKT-dependent direct and indirect regulation of cellular levels of reduced glutathione (GSH). Pharmacologic inhibition of specific AKT-dependent metabolic enzymes supporting defense against oxidative stress, e.g., inhibition of glutathione synthase and glutathione reductase, improved eradication of clonogenic tumor cells, particularly of TrC1 cells overexpressing AKT-E17K. We conclude that improved capacity of TrC1 AKT-E17K cells to balance antioxidant defense with provision of energy and other metabolites upon irradiation compared to TrC1 AKT-WT cells contributes to their increased radiation resistance. Our findings on the importance of glutathione de novo synthesis and glutathione regeneration for radiation resistance of TrC1 AKT-E17K cells offer novel perspectives for improving radiosensitivity in cancer cells with aberrant AKT activity by combining IR with inhibitors targeting AKT-dependent regulation of GSH provision.

Hypoxia aggravates ferroptosis in RPE cells by promoting the Fenton reaction.

Oxidative stress and hypoxia in the retinal pigment epithelium (RPE) have long been considered major risk factors in the pathophysiology of age-related macular degeneration (AMD), but systematic investigation of the interplay between these two risk factors was lacking. For this purpose, we treated a human RPE cell line (ARPE-19) with sodium iodate (SI), an oxidative stress agent, together with dimethyloxalylglycine (DMOG) which leads to stabilization of hypoxia-inducible factors (HIFs), key regulators of cellular adaptation to hypoxic conditions. We found that HIF stabilization aggravated oxidative stress-induced cell death by SI and iron-dependent ferroptosis was identified as the main cell death mechanism. Ferroptotic cell death depends on the Fenton reaction where H2O2 and iron react to generate hydroxyl radicals which trigger lipid peroxidation. Our findings clearly provide evidence for superoxide dismutase (SOD) driven H2O2 production fostering the Fenton reaction as indicated by triggered SOD activity upon DMOG + SI treatment as well as by reduced cell death levels upon SOD2 knockdown. In addition, iron transporters involved in non-transferrin-bound Fe2+ import as well as intracellular iron levels were also upregulated. Consequently, chelation of Fe2+ by 2'2-Bipyridyl completely rescued cells. Taken together, we show for the first time that HIF stabilization under oxidative stress conditions aggravates ferroptotic cell death in RPE cells. Thus, our study provides a novel link between hypoxia, oxidative stress and iron metabolism in AMD pathophysiology. Since iron accumulation and altered iron metabolism are characteristic features of AMD retinas and RPE cells, our cell culture model is suitable for high-throughput screening of new treatment approaches against AMD.

Metabolism of cancer cells commonly responds to irradiation by a transient early mitochondrial shutdown.

Cancer bioenergetics fuel processes necessary to maintain viability and growth under stress conditions. We hypothesized that cancer metabolism supports the repair of radiation-induced DNA double-stranded breaks (DSBs). We combined the systematic collection of metabolic and radiobiological data from a panel of irradiated cancer cell lines with mathematical modeling and identified a common metabolic response with impact on the DSB repair kinetics, including a mitochondrial shutdown followed by compensatory glycolysis and resumption of mitochondrial function. Combining ionizing radiation (IR) with inhibitors of the compensatory glycolysis or mitochondrial respiratory chain slowed mitochondrial recovery and DNA repair kinetics, offering an opportunity for therapeutic intervention. Mathematical modeling allowed us to generate new hypotheses on general and individual mechanisms of the radiation response with relevance to DNA repair and on metabolic vulnerabilities induced by cancer radiotherapy. These discoveries will guide future mechanistic studies for the discovery of metabolic targets for overcoming intrinsic or therapy-induced radioresistance.

Metabolic reprograming of antioxidant defense: a precision medicine perspective for radiotherapy of lung cancer?

Radiotherapy plays a key role in the management of lung cancer patients in curative and palliative settings. Traditionally, radiotherapy was either given alone or in combination with surgery, classical cytotoxic chemotherapy, or both. Technical and physical innovations achieved during the last two decades have helped to enhance the accuracy of radiotherapy dose delivery and have facilitated geometric radiotherapy individualization. Furthermore, multimodal combinations with molecularly tailored drugs or immunotherapy yielded promising survival benefits in selected patients. Yet high locoregional failure rates and frequent development of metastases still limit the patient outcome. One major obstacle to successful treatment is the high molecular heterogeneity observed in lung cancer. So far, clinical radiotherapy does not routinely use the knowledge on molecular subtypes with regard to therapy individualization and predictive biomarkers are missing. Herein, altered cancer metabolism has attracted novel attention during recent years as it promotes tumor growth and progression as well as resistance to anticancer therapies. The present perspective will exemplarily highlight how clinically relevant molecular subtypes defined by co-occurring somatic mutations in KRAS-driven lung cancer impact the metabolic phenotype of cancer cells, how the metabolic phenotype supports intrinsic radioresistance by the improved antioxidant defense, and also discuss potential subtype-specific actionable metabolic vulnerabilities. Understanding metabolic phenotypes of radioresistance and metabolic bottlenecks of cancer cells undergoing radiotherapy in a cancer-specific context will offer largely unexploited future avenues for biological individualization and optimization of radiotherapy. Transcriptional profiles will provide additional benefit in defining metabolic phenotypes associated with radioresistance, particularly in cases, where such dependencies cannot be identified by specific somatic mutations.