LAMTOR/Ragulator regulates lipid metabolism in macrophages and foam cell differentiation.

Late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) is a scaffold protein complex that anchors and regulates multiprotein signaling units on late endosomes/lysosomes. To identify LAMTOR-modulated endolysosomal proteins, primary macrophages were derived from bone marrow of conditional knockout mice carrying a specific deletion of LAMTOR2 in the monocyte/macrophage cell lineage. Affymetrix-based transcriptomic analysis and quantitative iTRAQ-based organelle proteomic analysis of endosomes derived from macrophages were performed. Further analyses showed that LAMTOR could be a novel regulator of foam cell differentiation. The lipid droplet formation phenotype observed in macrophages was additionally confirmed in MEFs, where lipidomic analysis identified cholesterol esters as specifically downregulated in LAMTOR2 knockout cells. The data obtained indicate a function of LAMTOR2 in lipid metabolism.

Biogenesis of lysosome-related organelles complex-1 (BORC) regulates late endosomal/lysosomal size through PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate.

Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC-1-related complex (BORC) regulates the size of these organelles via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2 ] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P2 , suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP-activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen-activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.

LAMTOR2 regulates dendritic cell homeostasis through FLT3-dependent mTOR signalling.

The receptor tyrosine kinase Flt3 and its ligand are crucial for dendritic cell (DC) homeostasis by activating downstream effectors including mammalian target of Rapamycin (mTOR) signalling. LAMTOR2 is a member of the Ragulator/LAMTOR complex known to regulate mTOR and extracellular signal-regulated kinase activation on the late endosome as well as endosomal biogenesis. Here we show in mice that conditional ablation of LAMTOR2 in DCs results in a severe disturbance of the DC compartment caused by accumulation of Flt3 on the cell surface. This results in an increased downstream activation of the AKT/mTOR signalling pathway and subsequently to a massive expansion of conventional DCs and plasmacytoid DCs in ageing mice. Finally, we can revert the symptoms in vivo by inhibiting the activation of Flt3 and its downstream target mTOR.

Glioma cell death induced by irradiation or alkylating agent chemotherapy is independent of the intrinsic ceramide pathway.

BACKGROUND/AIMS: Resistance to genotoxic therapy is a characteristic feature of glioma cells. Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and glucosylceramide synthase (GCS) catalyzes ceramide metabolism. Increased ceramide levels have been suggested to enhance chemotherapy-induced death of cancer cells. METHODS: Microarray and clinical data for ASM and GCS in astrocytomas WHO grade II-IV were acquired from the Rembrandt database. Moreover, the glioblastoma database of the Cancer Genome Atlas network (TCGA) was used for survival data of glioblastoma patients. For in vitro studies, increases in ceramide levels were achieved either by ASM overexpression or by the GCS inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) in human glioma cell lines. Combinations of alkylating chemotherapy or irradiation and ASM overexpression, PPMP or exogenous ceramide were applied in parental cells. The anti-glioma effects were investigated by assessing proliferation, metabolic activity, viability and clonogenicity. Finally, viability and clonogenicity were assessed in temozolomide (TMZ)-resistant cells upon treatment with PPMP, exogenous ceramide, alkylating chemotherapy, irradiation or their combinations. RESULTS: Interrogations from the Rembrandt and TCGA database showed a better survival of glioblastoma patients with low expression of ASM or GCS. ASM overexpression or PPMP treatment alone led to ceramide accumulation but did not enhance the anti-glioma activity of alkylating chemotherapy or irradiation. PPMP or exogenous ceramide induced acute cytotoxicity in glioblastoma cells. Combined treatments with chemotherapy or irradiation led to additive, but not synergistic effects. Finally, no synergy was found when TMZ-resistant cells were treated with exogenous ceramide or PPMP alone or in combination with TMZ or irradiation. CONCLUSION: Modulation of intrinsic glioma cell ceramide levels by ASM overexpression or GCS inhibition does not enhance the anti-glioma activity of alkylating chemotherapy or irradiation.

Monitoring the glioma tropism of bone marrow-derived progenitor cells by 2-photon laser scanning microscopy and positron emission tomography.

Intracerebral experimental gliomas attract intravenously injected murine or human bone marrow-derived hematopoietic progenitor and stem cells (HPC) in vitro, ex vivo, and in vivo, indicating that these progenitor cells might be suitable vehicles for a cell-based delivery of therapeutic molecules to malignant gliomas. With regard to therapeutic application, it is important to investigate cell fates in vivo (i.e., the time-dependent intratumoral and systemic distribution after intravenously injection). Conventional histological analysis has limitations in this regard because longitudinal monitoring is precluded. Here, we used 2-photon laser scanning microscopy (2PLSM), positron emission tomography (PET), and MRI to study the fate of intravenously injected HPC carrying fluorescence, bioluminescence, and PET reporter genes in glioma-bearing mice. Our 2PLSM-based monitoring studies revealed that HPC homing to intracerebral experimental gliomas occurred already within the first 6 h and was most efficient within the first 24 h after intravenous injection. The highest PET signals were detected in intracerebral gliomas, whereas the tracer uptake in other organs, notably spleen, lung, liver, and muscle, remained at background levels. The results have important implications for designing schedules for therapeutic cell-based anti-glioma approaches. Moreover, the PET reporter-based imaging technique will allow noninvasive monitoring of cell fate in future cell-based therapeutic antiglioma approaches.

Glioma tropism of lentivirally transduced hematopoietic progenitor cells.

Experimental gliomas attract hematopoietic progenitor cells (HPC) in vivo. HPC are therefore promising candidates for a cell-based delivery of therapeutic molecules to experimental gliomas. A therapeutic application requires efficient genetic manipulation of the cellular vector and a lack of tumorigenicity. Here, we studied the impact of lenti-viral transduction on the glioma tropism of human or murine HPC. Transduction of human or murine HPC with a GFP lentivirus (lenti-GFP) did not interfere with the glioma-mediated attraction of HPC. Bone marrow reconstitution of C57Bl/6 mice with syngeneic GFP-transgenic lineage-depleted bone marrow cells (lin- BM) was as efficient as reconstitution with syngeneic lin- BM transduced ex vivo with lenti-GFP. SMA-560 gliomas growing orthotopically in lenti-GFP-reconstituted VM/Dk mice recruited GFP-positive bone marrow-derived cells. Thus, lentiviral transduction did not interfere with the attraction of exogenously injected HPC or endogenous bone marrow-derived cells by experimental gliomas. Lenti-GFP-HPC implanted directly into tumor-free brains were not tumorigenic. The intravenous injection of lenti-GFP-HPC in glioma-bearing mice did not alter the survival of otherwise untreated animals and had no impact on the survival benefit conferred by cerebral irradiation. Taken together, genetic manipulation of HPC with lenti-GFP neither made these cells tumorigenic nor interfered with their glioma tropism.

SC68896, a novel small molecule proteasome inhibitor, exerts antiglioma activity in vitro and in vivo.

PURPOSE: Glioblastomas are among the most lethal neoplasms, with a median survival of <1 year. Modulation of the proteasome function has emerged as a novel approach to cancer pharmacotherapy. Here, we characterized the antitumor properties of SC68896, a novel small molecule proteasome inhibitor. EXPERIMENTAL DESIGN: Different tumor cell lines were tested by crystal violet staining for sensitivity to SC68896, given alone or in combination with death ligands. The molecular mechanisms mediating SC68896-induced cell death and changes in cell cycle progression were assessed by immunoblot and flow cytometry. An orthotopic human glioma xenograft model in nude mice was used to examine the in vivo activity of SC68896. RESULTS: SC68896 inhibits the proliferation of cell lines of different types of cancer, including malignant glioma. Exposure of LNT-229 glioma cells to SC68896 results in a concentration- and time-dependent inhibition of the proteasome, with a consequent accumulation of p21 and p27 proteins, cell cycle arrest, caspase cleavage, and induction of apoptosis. Using RNA interference, we show that the effect of SC68896 on glioma cells is facilitated by wild-type p53. SC68896 sensitizes glioma cells to tumor necrosis factor-related apoptosis-inducing ligand and CD95 ligand and up-regulates the cell surface expression of the tumor necrosis factor-related apoptosis-inducing ligand receptor cell death receptors 4 and 5, which may contribute to this sensitization. Intracerebral glioma-bearing nude mice treated either i.p. or intratumorally with SC68896 experience prolonged survival. CONCLUSIONS: SC68896 is the first proteasome inhibitor that exerts antiglioma activity in vivo. It may represent a novel prototype agent for the treatment of malignant gliomas and warrants clinical evaluation.

VEGF-dependent induction of CD62E on endothelial cells mediates glioma tropism of adult haematopoietic progenitor cells.

Haematopoietic progenitor cells (HPC) are attracted by experimental gliomas in vivo. This attraction is further enhanced by irradiation or hypoxic preconditioning of the glioma cells. Adhesive interactions might be critical to the preferential accumulation of HPC within the glioma tissue. Here, we studied the interactions of HPC with endothelial cells. Exposure of human cerebral endothelial cells (SV-HCEC), human microvascular endothelial cells (HMEC) and brain tumour endothelial cells derived from human glioblastomas (BTEC) to supernatants of glioma cells and primary glioma cells (SN-G) induced the expression of E-selectin (CD62E). CD62E expression was further enhanced when the glioma cells had been exposed to irradiation or hypoxia prior to the collection of supernatants, as well as by irradiation or exposure to hypoxia of the endothelial cells. Vascular cell adhesion molecule 1 (VCAM-1) was constitutively expressed on SV-HCEC, HMEC and BTEC, but was not modulated by SN-G, irradiation or hypoxia. Transendothelial HPC migration was enhanced after CD62E induction in vitro. Neutralizing antibodies to CD62E strongly reduced the homing of lin(-)Sca-1(+)c-kit(+) cells to orthotopic SMA-560 gliomas in vivo. Tissue microarray sampling normal brain tissue and astrocytomas of WHO grades II-IV revealed a selective expression of CD62E on endothelial cells of tumour vessels. SN-G-induced CD62E expression on endothelial cells in vitro required transforming growth factor (TGF)-beta signalling in glioma cells and vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGF-R2) signalling in endothelial cells. Further, we observed a nuclear factor kappa B-dependent activation of the CD62E promoter peaking at 12 h after VEGF-R2 activation by glioma-derived VEGF. Taken together, we identify glioma cell-induced CD62E expression on endothelial cells as one mediator of the glioma tropism of HPC.