Targeting of Deregulated Wnt/ß-Catenin Signaling by PRI-724 and LGK974 Inhibitors in Germ Cell Tumor Cell Lines.

The majority of patients with testicular germ cell tumors (GCTs) can be cured with cisplatin-based chemotherapy. However, for a subset of patients present with cisplatin-refractory disease, which confers a poor prognosis, the treatment options are limited. Novel therapies are therefore urgently needed to improve outcomes in this challenging patient population. It has previously been shown that Wnt/ß-catenin signaling is active in GCTs suggesting that its inhibitors LGK974 and PRI-724 may show promise in the management of cisplatin-refractory GCTs. We herein investigated whether LGK-974 and PRI-724 provide a treatment effect in cisplatin-resistant GCT cell lines. Taking a genoproteomic approach and utilizing xenograft models we found the increased level of ß-catenin in 2 of 4 cisplatin-resistant (CisR) cell lines (TCam-2 CisR and NCCIT CisR) and the decreased level of ß-catenin and cyclin D1 in cisplatin-resistant NTERA-2 CisR cell line. While the effect of treatment with LGK974 was limited or none, the NTERA-2 CisR exhibited the increased sensitivity to PRI-724 in comparison with parental cell line. Furthermore, the pro-apoptotic effect of PRI-724 was documented in all cell lines. Our data strongly suggests that a Wnt/ß-catenin signaling is altered in cisplatin-resistant GCT cell lines and the inhibition with PRI-724 is effective in NTERA-2 CisR cells. Further evaluation of Wnt/ß-catenin pathway inhibition in GCTs is therefore warranted.

Classic and targeted anti-leukaemic agents interfere with the cholesterol biogenesis metagene in acute myeloid leukaemia: Therapeutic implications.

Despite significant advances in deciphering the molecular landscape of acute myeloid leukaemia (AML), therapeutic outcomes of this haematological malignancy have only modestly improved over the past decades. Drug resistance and disease recurrence almost invariably occur, highlighting the need for a deeper understanding of these processes. While low O2 compartments, such as bone marrow (BM) niches, are well-recognized hosts of drug-resistant leukaemic cells, standard in vitro studies are routinely performed under supra-physiologic (21% O2 , ambient air) conditions, which limits clinical translatability. We hereby identify molecular pathways enriched in AML cells that survive acute challenges with classic or targeted therapeutic agents. Experiments took into account variations in O2 tension encountered by leukaemic cells in clinical settings. Integrated RNA and protein profiles revealed that lipid biosynthesis, and particularly the cholesterol biogenesis branch, is a particularly therapy-induced vulnerability in AML cells under low O2 states. We also demonstrate that the impact of the cytotoxic agent cytarabine is selectively enhanced by a high-potency statin. The cholesterol biosynthesis programme is amenable to additional translational opportunities within the expanding AML therapeutic landscape. Our findings support the further investigation of higher-potency statin (eg rosuvastatin)-based combination therapies to enhance targeting residual AML cells that reside in low O2 environments.

Effects of lipid interactions on model vesicle engulfment by alveolar macrophages.

The engulfment function of macrophages relies on complex molecular interactions involving both lipids and proteins. In particular, the clearance of apoptotic bodies (efferocytosis) is enabled by externalization on the cell target of phosphatidylserine lipids, which activate receptors on macrophages, suggesting that (local) specific lipid-protein interactions are required at least for the initiation of efferocytosis. However, in addition to apoptotic cells, macrophages can engulf foreign bodies that vary substantially in size from a few nanometers to microns, suggesting that nonspecific interactions over a wide range of length scales could be relevant. Here, we use model lipid membranes (made of phosphatidylcholine, phosphatidylserine, and ceramide) and rat alveolar macrophages to show how lipid bilayer properties probed by small-angle x-ray scattering and solid-state (2)H NMR correlate with engulfment rates measured by flow cytometry. We find that engulfment of protein-free model lipid vesicles is promoted by the presence of phosphatidylserine lipids but inhibited by ceramide, in accord with a previous study of apoptotic cells. We conclude that the roles of phosphatidylserine and ceramide in phagocytosis is based, at least in part, on lipid-mediated modification of membrane physical properties, including interactions at large length scales as well as local lipid ordering and possible domain formation.

Smoking exposure induces human lung endothelial cell adaptation to apoptotic stress.

Prolonged exposure to cigarette smoking is the main risk factor for emphysema, a component of chronic obstructive pulmonary diseases (COPDs) characterized by destruction of alveolar walls. Moreover, smoking is associated with pulmonary artery remodeling and pulmonary hypertension, even in the absence of COPD, through as yet unexplained mechanisms. In murine models, elevations of intra- and paracellular ceramides in response to smoking have been implicated in the induction of lung endothelial cell apoptosis, but the role of ceramides in human cell counterparts is yet unknown. We modeled paracrine increases (outside-in) of palmitoyl ceramide (Cer16) in primary human lung microvascular cells. In naive cells, isolated from nonsmokers, Cer16 significantly reduced cellular proliferation and induced caspase-independent apoptosis via mitochondrial membrane depolarization, apoptosis-inducing factor translocation, and poly(ADP-ribose) polymerase cleavage. In these cells, caspase-3 was inhibited by ceramide-induced Akt phosphorylation, and by the induction of autophagic microtubule-associated protein-1 light-chain 3 lipidation. In contrast, cells isolated from smokers exhibited increased baseline proliferative features associated with lack of p16(INK4a) expression and Akt hyperphosphorylation. These cells were resistant to Cer16-induced apoptosis, despite presence of both endoplasmic reticulum stress response and mitochondrial membrane depolarization. In cells from smokers, the prominent up-regulation of Akt pathways inhibited ceramide-triggered apoptosis, and was associated with elevated sphingosine and high-mobility group box 1, skewing the cell's response toward autophagy and survival. In conclusion, the cell responses to ceramide are modulated by an intricate cross-talk between Akt signaling and sphingolipid metabolites, and profoundly modified by previous cigarette smoke exposure, which selects for an apoptosis-resistant phenotype.

Solid-state ²H NMR shows equivalence of dehydration and osmotic pressures in lipid membrane deformation.

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state ²H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our ²H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S(CD)) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state ²H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state ²H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.