No abstract

Here, we developed a new synthetic lethal strategy for further optimizing the eradication of cancer stem cells (CSCs). Briefly, we show that chronic treatment with the FDA-approved antibiotic Doxycycline effectively reduces cellular respiration, by targeting mitochondrial protein translation. The expression of four mitochondrial DNA encoded proteins (MT-ND3, MT-CO2, MT-ATP6 and MT-ATP8) is suppressed, by up to 35-fold. This high selection pressure metabolically synchronizes the surviving cancer cell sub-population towards a predominantly glycolytic phenotype, resulting in metabolic inflexibility. We directly validated this Doxycycline-induced glycolytic phenotype, by using metabolic flux analysis and label-free unbiased proteomics. Next, we identified two natural products (Vitamin C and Berberine) and six clinically-approved drugs, for metabolically targeting the Doxycycline-resistant CSC population (Atovaquone, Irinotecan, Sorafenib, Niclosamide, Chloroquine, and Stiripentol). This new combination strategy allows for the more efficacious eradication of CSCs with Doxycycline, and provides a simple pragmatic solution to the possible development of Doxycycline-resistance in cancer cells. In summary, we propose the combined use of i) Doxycycline (Hit-1: targeting mitochondria) and ii) Vitamin C (Hit-2: targeting glycolysis), which represents a new synthetic-lethal metabolic strategy for eradicating CSCs. This type of metabolic Achilles' heel will allow us and others to more effectively "starve" the CSC population.

FTY720 is an immunosuppressant that reduces circulating levels of naïve lymphocytes by increasing their localization and sequestration in secondary lymphoid organs. It is considered to be an agonist for sphingosine 1-phosphate (S1P) G protein-coupled receptors (GPCRs) after phosphorylation at micromolar concentrations. We now describe its nonagonist and noncompetitive inhibitory activity at low nanomolar concentrations for types 1 and 5 S1P-GPCRs and of moderate potency for type 2 S1P-GPCRs. FTY720 blocks S1P signaling through S1P1,2,5 by inducing their internalization and intracellular partial degradation without affecting S1P3 or S1P4. S1P-R internalization is maximal several hours after only seconds of incubation with FTY720 at 37 degrees C and washing, and continues for days before recovery of surface expression and functions. The timing and extent of S1P-R internalization are highly dependent on FTY720 concentration. FTY720 is therefore an S1P-GPCR-selective and noncompetitive inhibitor with a unique mechanism of action.

The blood constituent sphingosine 1-phosphate (S1P) is a specific ligand for five G-protein-coupled receptors designated S1P(1-5). Expression of the S1P1 receptor on lymphocytes is required for their exit from secondary lymphoid organs, suggesting that S1P serves as a stimulus for maintaining lymphocyte circulation in blood. Despite its potential role in immune surveillance, the regulatory system that controls blood S1P levels is not well understood. This report reveals that erythrocytes constitute a buffer system for S1P in blood. They efficiently incorporated and stored S1P, and protected it from cellular degradation. They also released S1P into plasma, but not into other serum-free media, indicating that S1P release was controlled by a plasma factor. Erythrocytes did not generate S1P since an increase in plasma S1P levels was always accompanied by a decrease in cellular S1P levels. Thrombocytes that were reported to generate and release S1P after activation did not contribute to the observed S1P release in blood. The amount of erythrocytes as well as the proportion of plasma in the medium determined the magnitude of S1P release. Adoptively transferred S1P-loaded and unloaded mouse erythrocytes displayed a normal life span and similar S1P levels 24 h after recovery, indicating that S1P incorporation and release are dynamically regulated in vivo.