Glucose Limitation Sensitizes Cancer Cells to Selenite-Induced Cytotoxicity via SLC7A11-Mediated Redox Collapse

Combination of intermittent fasting and chemotherapy has been drawn an increasing attention because of the encouraging efficacy. In this study, we evaluated the anti-cancer effect of combination of glucose limitation and selenite (Se), a representative inorganic form of selenium, that is preferentially accumulated in tumors. Results showed that cytotoxic effect of selenite on cancer cells, but not on normal cells, was significantly enhanced in response to the combination of selenite and glucose limitation. Furthermore, in vivo therapeutic efficacy of combining selenite with fasting was dramatically improved in xenograft models of lung and colon cancer. Mechanistically, we found that SLC7A11 expression in cancer cells was up-regulated by selenite both in vitro and in vivo. The elevated SLC7A11 led to cystine accumulation, NADPH depletion and the conversion of cystine to cysteine inhibition, which in turn boosted selenite-mediated reactive oxygen species (ROS), followed by enhancement of selenite-mediated cytotoxic effect. The findings of the present study provide an effective and practical approach for increasing the therapeutic window of selenite and imply that combination of selenite and fasting holds promising potential to be developed a clinically useful regimen for treating certain types of cancer.

The sensor histidine kinase ArlS is necessary for Staphylococcus aureus to activate ArlR in response to nutrient availability

Staphylococcus aureus is a versatile opportunistic pathogen whose success is driven by its ability to adapt to diverse environments and host-imposed stresses. Two-component signal transduction systems, such as ArlRS, often mediate these adaptations. Loss of ArlRS or the response regulator ArlR alone impairs the ability of S. aureus to respond to host-imposed manganese starvation and glucose limitation. As sensor histidine kinases and response regulators frequently work as pairs, it has been assumed that ArlS senses and activates ArlR in response to these stimuli. However, recent work suggests that the sensor histidine kinase GraS can also activate ArlR, calling the contribution of ArlS in responding to manganese and glucose availability into question. The current studies reveal that ArlS is necessary to activate ArlR in response to manganese sequestration by the host immune effector calprotectin and glucose limitation. Although the loss of ArlS does not completely eliminate ArlR activity, this response regulator is no longer responsive to manganese or glucose availability in the absence of its cognate histidine kinase. Despite the residual activity of ArlR in the absence of ArlS, ArlR phosphorylation by ArlS is required for S. aureus to resist calprotectin-imposed metal starvation. Cumulatively, these findings contribute to the understanding of S. aureus signaling transduction in response to nutritional immunity and support the previous observation that indicates ArlRS is activated by a common signal derived from host-imposed manganese and glucose limitation. IMPORTANCE The ability of pathogens, including Staphylococcus aureus , to sense and adapt to diverse environments partially relies on two-component systems, such as ArlRS. Recent work revealed that the response regulator ArlR can be cross-activated by the sensor histidine kinase GraS, rendering the role of its cognate partner, ArlS, in response to manganese and glucose limitation uncertain. This study reveals that ArlS is necessary for the activation of ArlR in response to calprotectin and glucose limitation. Although a low level of ArlR activity remains in the absence of ArlS, ArlS phosphotransfer to ArlR is required for S. aureus to overcome calprotectin-induced nutritional stress. Collectively, this study provides fundamental information to understand how ArlRS mediates staphylococcal adaptation during infection.

Phosphatase PHLPP2 regulates the cellular response to metabolic stress through AMPK

PHLPP2 is a member of the PHLPP family of phosphatases, known to suppress cell growth by inhibiting proliferation or promoting apoptosis. Oncogenic kinases Akt, S6K, and PKC, and pro-apoptotic kinase Mst1, have been recognized as functional targets of the PHLPP family. However, we observed that, in T-leukemia cells subjected to metabolic stress from glucose limitation, PHLPP2 specifically targets the energy-sensing AMP-activated protein kinase, pAMPK, rather than Akt or S6K. PHLPP2 dephosphorylates pAMPK in several other human cancer cells as well. PHLPP2 and pAMPK interact with each other, and the pleckstrin homology (PH) domain on PHLPP2 is required for their interaction, for dephosphorylating and inactivating AMPK, and for the apoptotic response of the leukemia cells to glucose limitation. Silencing PHLPP2 protein expression prolongs the survival of leukemia cells subjected to severe glucose limitation by promoting a switch to AMPK-mediated fatty acid oxidation for energy generation. Thus, this study reveals a novel role for PHLPP2 in suppressing a survival response mediated through AMPK signaling. Given the multiple ways in which PHLPP phosphatases act to oppose survival signaling in cancers and the pivotal role played by AMPK in redox homeostasis via glucose and fatty acid metabolism, the revelation that AMPK is a target of PHLPP2 could lead to better therapeutics directed both at cancer and at metabolic diseases.

Glucose Limitation Sensitizes Cancer Cells to Selenite-Induced Cytotoxic Effect via SLC7A11-Mediated Redox Collapse

Background: Combination of fasting with chemotherapy has been drawn an increasing attention because of the encouraging efficacy. SLC7A11 is frequently over-expressed in most of cancer cells, and elevated expression of SLC7A11 renders cancer cells more susceptible to glucose starvation owing to SLC7A11-mediated redox collapse. Selenite is a representative inorganic form of selenium, and is preferentially accumulated in tumors. This selenophilic peculiarity of cancer cells is closely associated with the elevated expression of SLC7A11. Given the established the link among glucose deprivation, SLC7A11, oxidative stress and selenite, we hypothesized that glucose starvation could specifically sensitize cancer cells to selenite-mediated cytotoxic effect. Methods: The cytotoxic effect of combining selenite with glucose starvation on cancer cell was assessed by crystal violet staining and Annexin V/PI staining. Flow cytometry were employed to assess intracellular ROS levels, labile iron pool and lipid peroxidation. Xenograft models were used to test its in vivo antitumor activity. Commercial assay kit, LC-MS, RNA interference and western blot were applied to investigate the mechanism underlying synergistic effect.Results: It showed that cytotoxic effect of selenite on cancer cells, but not on normal cells, was significantly enhanced in response to the combination of selenite and glucose limitation. Furthermore, in vivo therapeutic efficacy of combining selenite with fasting was dramatically improved in xenograft models of lung and colon cancer. Mechanistically, we found that SLC7A11 expression in cancer cells was up-regulated by selenite both in vitro and in vivo. The elevated SLC7A11 led to accumulation of cystine, depletion of NADPH, and inhibition of cystine to cysteine conversion, which in turn boosted selenite-mediated reactive oxygen species (ROS), followed by enhancement of selenite-mediated cytotoxic effect. Conclusion: The findings of the present study provide an effective and practical approach for increasing the therapeutic window of selenite, and imply that combination of selenite with fasting holds promising potential to be developed a clinically useful regimen for treating certain types of cancer.

Glucose limitation activates AMPK coupled SENP1-Sirt3 signalling in mitochondria for T cell memory development

Metabolic programming and mitochondrial dynamics along with T cell differentiation affect T cell fate and memory development; however, how to control metabolic reprogramming and mitochondrial dynamics in T cell memory development is unclear. Here, we provide evidence that the SUMO protease SENP1 promotes T cell memory development via Sirt3 deSUMOylation. SENP1-Sirt3 signalling augments the deacetylase activity of Sirt3, promoting both OXPHOS and mitochondrial fusion. Mechanistically, SENP1 activates Sirt3 deacetylase activity in T cell mitochondria, leading to reduction of the acetylation of mitochondrial metalloprotease YME1L1. Consequently, deacetylation of YME1L1 suppresses its activity on OPA1 cleavage to facilitate mitochondrial fusion, which results in T cell survival and promotes T cell memory development. We also show that the glycolytic intermediate fructose-1,6-bisphosphate (FBP) as a negative regulator suppresses AMPK-mediated activation of the SENP1-Sirt3 axis and reduces memory development. Moreover, glucose limitation reduces FBP production and activates AMPK during T cell memory development. These data show that glucose limitation activates AMPK and the subsequent SENP1-Sirt3 signalling for T cell memory development.

Yeast cells under glucose-limitation environment need increased response cost for osmostress defense

Cells always make responses to environmental changes, involving dynamic expression of tens to hundreds of proteins. This response system may demand substantial cost and thus affect cell growth. Here, we studied the cell's responses to osmostress under glucose-limitation environments. Through analyzed thirteen osmotic-downstream proteins and two related transcription factors, we found that the cells required stronger responses under low glucose concentrations than normal glucose condition after being stimulated by osmostress, even the cell growth rate was unchanged in these two constant conditions. We proposed and verified that under a glucose-limitation environment, the glycolysis intermediates were limited (defense reserve saving), which caused that cells needed more glycerol production enzymes to adapt to the osmostress. Further experiments proved that this 'defense reserve-saving' strategy required cells to spend more response cost when facing stress, which on the other hand, enhanced the fitness for the coming environment variations via protein accumulation reserve.

Whole-cell modeling in yeast predicts compartment-specific proteome constraints that drive metabolic strategies

When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource re-allocation under cellular constraints. Eukaryal cells contain metabolically active organelles such as mitochondria, competing for cytosolic space and resources, and the nature of the relevant cellular constraints remain to be determined for such cells. Here we developed a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions (16304 reactions in total). The model predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate. Comparing model predictions with quantitative experimental data revealed that under glucose limitation, a mitochondrial constraint limits growth at the onset of ethanol formation - known as the Crabtree effect. Under sugar excess, however, a constraint on total cytosolic volume dictates overflow metabolism. Our comprehensive model thus identifies condition-dependent and compartment-specific constraints that can explain metabolic strategies and protein expression profiles from growth rate optimization, providing a framework to understand metabolic adaptation in eukaryal cells.

Encystation of Entamoeba histolytica in Axenic Culture

Entamoeba histolytica is a parasitic protozoan that causes amoebic dysentery, which affects approximately 90 million people each year worldwide. E. histolytica is transmitted through ingestion of food and water contaminated with the cyst form, which undergoes excystation in the small intestine to the trophozoite form that colonizes the large intestine. The reptile pathogen Entamoeba invadens has served as a model for studying stage conversion between the trophozoite and cyst form due to lack of reproducible encystation of E. histolytica in the laboratory. Although much has been learned about encystation and excystation using E. invadens, the findings do not fully translate to E. histolytica due to the extensive genetic and host differences between these species. Here, we present the first reproducible encystation of E. histolytica in vitro. The cysts produced were viable and displayed the four characteristic hallmarks: round shape, chitinous cell wall, tetranucleation, and detergent resistance. Using flow cytometry analysis, glucose limitation and high cell density were key for encystation, as for E. invadens. Entry into encystation was enhanced by the short-chain fatty acids acetate and propionate, unlike for E. invadens. This new model will now allow the further study of E. histolytica stage conversion, transmission, and treatment.

Fasting reverses drug-resistance in hepatocellular carcinoma through p53-dependent metabolic synergism

ABSTRACTCancer cells voraciously consume nutrients to support their growth, exposing a metabolic vulnerability that can be therapeutically exploited. Here we show in hepatocellular carcinoma (HCC) cells, xenografts, and in patient-derived HCC organoids that fasting can synergistically sensitize resistant HCC to sorafenib. Mechanistically, sorafenib acts non-canonically as an inhibitor of mitochondrial respiration, causing resistant cells to depend on glycolysis for survival. Fasting, through reduction in glucose and impeded AKT/mTOR-signaling, prevents this Warburg shift. Regulating glucose transporter and pro-apoptotic protein expression, p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting. p53 is also crucial for fasting-mediated improvement of sorafenib efficacy in an orthotopic HCC mouse model. Together, our data suggest intermittent fasting and sorafenib as rational combination therapy for HCC with intact p53 signaling. As HCC therapy is currently severely limited by resistance, these results should instigate clinical studies aimed at improving therapy response in advanced-stage, and possibly even early-stage, HCC.HIGHLIGHTSFasting sensitizes resistant HCC xenografts and patient-derived organoids to sorafenibSorafenib-mediated Warburg shift is prevented by glucose limitation upon fastingFasting synergistically improves sorafenib efficacy in non-resistant modelsp53 is required for synergism by regulating glucose uptake and apoptosisGRAPHICAL ABSTRACT

Carbon source-dependent reprogramming of anaerobic metabolism in Staphylococcus aureus

To be a successful pathogen, S. aureus has to adapt its metabolism to the typically oxygen- and glucose-limited environment of the host. Under fermenting conditions and in the presence of glucose, S. aureus uses glycolysis to generate ATP via substrate level phosphorylation and mainly lactic acid fermentation to maintain the redox balance by re-oxidation of NADH equivalents. However, it is less clear how S. aureus proceeds under anoxic conditions and glucose limitation, likely representing the bona-fide situation in the host. Using a combination of proteomic, transcriptional and metabolomic analyses, we show that in the absence of an abundant glycolysis substrate the available carbon source pyruvate is converted to acetyl-CoA (AcCoA) in a pyruvate formate-lyase (PflB)-dependent reaction to produce ATP and acetate. This process critically depends on de-repression of the catabolite control protein A (CcpA), leading to upregulation of pflB transcription. Under these conditions, ethanol production is repressed to prevent wasteful consumption of AcCoA. In addition, our global and quantitative characterization of the metabolic switch prioritizing acetate over lactate fermentation when glucose is absent illustrates examples of carbon source-dependent control of colonization and pathogenicity factors. Importance: Under infection conditions, S. aureus needs to ensure survival when energy production via oxidative phosphorylation is not possible, e.g. either due to the lack of terminal electron acceptors or by the inactivation of components of the respiratory chain. Under these conditions, S. aureus can switch to mixed acid fermentation to sustain ATP production by substrate-level phosphorylation. The drop in the cellular NAD+/NADH ratio is sensed by the repressor Rex, resulting in de-repression of fermentation genes. Here we show that expression of fermentation pathways is further controlled by CcpA in response to the availability of glucose to ensure optimal resource utilization under growth limiting conditions. We provide evidence for carbon source-dependent control of colonization and virulence factors. These findings add another level to the regulatory network controlling mixed acid fermentation in S. aureus and provide additional evidence for the lifestyle-modulating effect of carbon sources available in S. aureus.