p66ShcA promotes malignant breast cancer phenotypes by alleviating energetic and oxidative stress.

Significant efforts have focused on identifying targetable genetic drivers that support the growth of solid tumors and/or increase metastatic ability. During tumor development and progression to metastatic disease, physiological and pharmacological selective pressures influence parallel adaptive strategies within cancer cell sub-populations. Such adaptations allow cancer cells to withstand these stressful microenvironments. This Darwinian model of stress adaptation often prevents durable clinical responses and influences the emergence of aggressive cancers with increased metastatic fitness. However, the mechanisms contributing to such adaptive stress responses are poorly understood. We now demonstrate that the p66ShcA redox protein, itself a ROS inducer, is essential for survival in response to physiological stressors, including anchorage independence and nutrient deprivation, in the context of poor outcome breast cancers. Mechanistically, we show that p66ShcA promotes both glucose and glutamine metabolic reprogramming in breast cancer cells, to increase their capacity to engage catabolic metabolism and support glutathione synthesis. In doing so, chronic p66ShcA exposure contributes to adaptive stress responses, providing breast cancer cells with sufficient ATP and redox balance needed to withstand such transient stressed states. Our studies demonstrate that p66ShcA functionally contributes to the maintenance of aggressive phenotypes and the emergence of metastatic disease by forcing breast tumors to adapt to chronic and moderately elevated levels of oxidative stress.

Alanine supplementation exploits glutamine dependency induced by SMARCA4/2-loss.

SMARCA4 (BRG1) and SMARCA2 (BRM) are the two paralogous ATPases of the SWI/SNF chromatin remodeling complexes frequently inactivated in cancers. Cells deficient in either ATPase have been shown to depend on the remaining counterpart for survival. Contrary to this paralog synthetic lethality, concomitant loss of SMARCA4/2 occurs in a subset of cancers associated with very poor outcomes. Here, we uncover that SMARCA4/2-loss represses expression of the glucose transporter GLUT1, causing reduced glucose uptake and glycolysis accompanied with increased dependency on oxidative phosphorylation (OXPHOS); adapting to this, these SMARCA4/2-deficient cells rely on elevated SLC38A2, an amino acid transporter, to increase glutamine import for fueling OXPHOS. Consequently, SMARCA4/2-deficient cells and tumors are highly sensitive to inhibitors targeting OXPHOS or glutamine metabolism. Furthermore, supplementation of alanine, also imported by SLC38A2, restricts glutamine uptake through competition and selectively induces death in SMARCA4/2-deficient cancer cells. At a clinically relevant dose, alanine supplementation synergizes with OXPHOS inhibition or conventional chemotherapy eliciting marked antitumor activity in patient-derived xenografts. Our findings reveal multiple druggable vulnerabilities of SMARCA4/2-loss exploiting a GLUT1/SLC38A2-mediated metabolic shift. Particularly, unlike dietary deprivation approaches, alanine supplementation can be readily applied to current regimens for better treatment of these aggressive cancers.

Autophagy-dependent glutaminolysis drives superior IL21 production in HIV-1-specific CD4 T cells.

The maintenance of a strong IL21 production in memory CD4 T cells, especially in HIV-1-specific cells, represents a major correlate of natural immune protection against the virus. However, the molecular mechanisms underlying IL21 production during HIV-1 infection, which is only elevated among the naturally protected elite controllers (EC), are still unknown. We recently found out that lipophagy is a critical immune mediator that control an antiviral metabolic state following CD8A T cell receptor engagement, playing an important role in the natural control of HIV-1 infection. This led us to investigate whether the beneficial role of a strong macroautophagy/autophagy, could also be used to ensure effective IL21 production as well. Herein, we confirm that after both polyclonal and HIV-1-specific activation, memory CD4 T cells (Mem) from EC display enhanced activity of the autophagy-mediated proteolysis compared to ART. Our results indicate that the enhanced autophagy activity in EC was controlled by the energy-sensing PRKAA1 (protein kinase AMP-activated catalytic subunit alpha 1). We further confirmed the critical role of the autophagy-mediated proteolysis in the strong IL21 production in EC by using BECN1 gene silencing as well as protease, PRKAA1, and lysosomal inhibitors. Finally, we established that high autophagy-mediated proteolysis in EC fuels their cellular rates of mitochondrial respiration due to glutaminolysis. Our data confirm the critical role of autophagy in dictating the metabolic input, which is required not only to ensure protective cytotoxic CD8A T cell responses, but also to provide strong IL21 production among antiviral CD4 T cells.Abbreviations: AKG: alpha-ketoglutarate; ART: patients under antiretroviral therapy; ATG7: autophagy related 7; BaF: bafilomycin A1; BECN1: beclin 1; Chloro.: chloroquine; EC: elite controllers; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FOXO3: forkhead box O3; GLS: glutaminase; GLUD1: glutamate dehydrogenase 1; HIVneg: HIV-1-uninfected control donors; IFNG/IFN-γ: interferon gamma; IL21: interleukin 21; MTOR: mechanistic target of rapamycin kinase; PBMC: peripheral blood mononuclear cells; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; SQSTM1: sequestosome 1; TCA: tricarboxylic acid cycle; ULK1: unc-51 like autophagy activating kinase.

Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Metabolic Adaptation and Enables Glucose-Independent Tumor Growth.

Cancer cells adapt metabolically to proliferate under nutrient limitation. Here we used combined transcriptional-metabolomic network analysis to identify metabolic pathways that support glucose-independent tumor cell proliferation. We found that glucose deprivation stimulated re-wiring of the tricarboxylic acid (TCA) cycle and early steps of gluconeogenesis to promote glucose-independent cell proliferation. Glucose limitation promoted the production of phosphoenolpyruvate (PEP) from glutamine via the activity of mitochondrial PEP-carboxykinase (PCK2). Under these conditions, glutamine-derived PEP was used to fuel biosynthetic pathways normally sustained by glucose, including serine and purine biosynthesis. PCK2 expression was required to maintain tumor cell proliferation under limited-glucose conditions in vitro and tumor growth in vivo. Elevated PCK2 expression is observed in several human tumor types and enriched in tumor tissue from non-small-cell lung cancer (NSCLC) patients. Our results define a role for PCK2 in cancer cell metabolic reprogramming that promotes glucose-independent cell growth and metabolic stress resistance in human tumors.

The complete targeted profile of the organic acid intermediates of the citric acid cycle using a single stable isotope dilution analysis, sodium borodeuteride reduction and selected ion monitoring GC/MS.

The quantitative profiling of the organic acid intermediates of the citric acid cycle (CAC) presents a challenge due to the lack of commercially available internal standards for all of the organic acid intermediates. We developed an analytical method that enables the quantitation of all the organic acids in the CAC in a single stable isotope dilution GC/MS analysis with deuterium-labeled analogs used as internal standards. The unstable α-keto acids are rapidly reduced with sodium borodeuteride to the corresponding stable α-deutero-α-hydroxy acids and these, along with their unlabeled analogs and other CAC organic acid intermediates, are converted to their tert-butyldimethylsilyl derivatives. Selected ion monitoring is employed with electron ionization. We validated this method by treating an untransformed mouse mammary epithelial cell line with well-known mitochondrial toxins affecting the electron transport chain and ATP synthase, which resulted in profound perturbations of the concentration of CAC intermediates.

Measurement of urinary trimethylamine and trimethylamime oxide by direct infusion electrospray quadrupole time-of-flight mass spectrometry.

Urinary trimethylamine (TMA) and its oxide (TMAOx) are measured separately and as a mixture using (15)N-labeled internal standards and direct infusion electrospray with a quadrupole time-of-flight (Q-ToF) instrument. TMA is quaternized with trideuteromethyl iodide to avoid inclusion of endogenous tetramethylammonium ion in the TMA measurement, whereas TMAOx is measured as the protonated molecule. Measurements reported as percentage TMA made with separate and combined samples agree within 6% of the measured values and demonstrate that both TMA and TMAOx can be measured simultaneously in a single analysis. Moreover, the analysis is simpler and less tedious and time-consuming than some earlier methods.