Research of Mitochondrial Function, Structure, Dynamics and Intracellular Organization.

Mitochondria have been recognized as the energy (in the form of ATP)-producing cell organelles, required for cell viability, survival and normal cell function [...].

Lengthening the Guanidine-Aryl Linker of Phenylpyrimidinylguanidines Increases Their Potency as Inhibitors of FOXO3-Induced Gene Transcription.

Increased FOXO3 nuclear localization is involved in neuroblastoma chemoresistance and tumor angiogenesis. Accordingly, FOXO3 inhibition is a promising strategy for boosting antitumor immune responses and suppressing FOXO3-mediated therapy resistance in cancer cells. However, no FOXO3 inhibitors are currently available for clinical use. Nevertheless, we have recently identified (4-propoxy)phenylpyrimidinylguanidine as a FOXO3 inhibitor in cancer cells in the low micromolar range. Here, we report the synthesis and structure-activity relationship study of a small library of its derivatives, some of which inhibit FOXO3-induced gene transcription in cancer cells in a submicromolar range and are thus 1 order of magnitude more potent than their parent compound. By NMR and molecular docking, we showed that these compounds differ in their interactions with the DNA-binding domain of FOXO3. These results may provide a foundation for further optimizing (4-propoxy)phenylpyrimidinylguanidine and developing therapeutics for inhibiting the activity of forkhead box (FOX) transcription factors and their interactions with other binding partners.

The Complex Interplay between Mitochondria, ROS and Entire Cellular Metabolism.

Besides their main function for energy production in form of ATP in processes of oxidative phosphorylation (OxPhos), mitochondria perform many other important cellular functions and participate in various physiological processes that are congregated. For example, mitochondria are considered to be one of the main sources of reactive oxygen species (ROS) and therefore they actively participate in the regulation of cellular redox and ROS signaling. These organelles also play a crucial role in Ca2+ signaling and homeostasis. The mitochondrial OxPhos and their cellular functions are strongly cell/tissue specific and can be heterogeneous even within the same cell, due to the existence of mitochondrial subpopulations with distinct functional and structural properties. However, the interplay between different functions of mitochondria is not fully understood. The mitochondrial functions may change as a response to the changes in the cellular metabolism (signaling in). On the other hand, several factors and feedback signals from mitochondria may influence the entire cell physiology (signaling out). Numerous interactions between mitochondria and the rest of cell, various cytoskeletal proteins, endoplasmic reticulum (ER) and other cellular elements have been demonstrated, and these interactions could actively participate in the regulation of mitochondrial and cellular metabolism. This review highlights the important role of the interplay between mitochondrial and entire cell physiology, including signaling from and to mitochondria.

3D bioprinted, vascularized neuroblastoma tumor environment in fluidic chip devices for precision medicine drug testing.

Neuroblastoma is an extracranial solid tumor which develops in early childhood and still has a poor prognosis. One strategy to increase cure rates is the identification of patient-specific drug responses in tissue models that mimic the interaction between patient cancer cells and tumor environment. We therefore developed a perfused and micro-vascularized tumor-environment model that is directly bioprinted into custom-manufactured fluidic chips. A gelatin-methacrylate/fibrin-based matrix containing multiple cell types mimics the tumor-microenvironment that promotes spontaneous micro-vessel formation by embedded endothelial cells. We demonstrate that both, adipocyte- and iPSC-derived mesenchymal stem cells can guide this process. Bioprinted channels are coated with endothelial cells post printing to form a dense vessel-tissue barrier. The tissue model thereby mimics structure and function of human soft tissue with endothelial cell-coated larger vessels for perfusion and micro-vessel networks within the hydrogel-matrix. Patient-derived neuroblastoma spheroids are added to the matrix during the printing process and grown for more than two weeks. We demonstrate that micro-vessels are attracted by and grow into tumor spheroids and that neuroblastoma cells invade the tumor-environment as soon as the spheroids disrupt. In summary, we describe the first bioprinted, micro-vascularized neuroblastoma-tumor-environment model directly printed into fluidic chips and a novel medium-throughput biofabrication platform suitable for studying tumor angiogenesis and metastasis in precision medicine approaches in future.

Analysis of Mitochondrial Function, Structure, and Intracellular Organization In Situ in Cardiomyocytes and Skeletal Muscles.

Analysis of the function, structure, and intracellular organization of mitochondria is important for elucidating energy metabolism and intracellular energy transfer. In addition, basic and clinically oriented studies that investigate organ/tissue/cell dysfunction in various human diseases, including myopathies, cardiac/brain ischemia-reperfusion injuries, neurodegenerative diseases, cancer, and aging, require precise estimation of mitochondrial function. It should be noted that the main metabolic and functional characteristics of mitochondria obtained in situ (in permeabilized cells and tissue samples) and in vitro (in isolated organelles) are quite different, thereby compromising interpretations of experimental and clinical data. These differences are explained by the existence of the mitochondrial network, which possesses multiple interactions between the cytoplasm and other subcellular organelles. Metabolic and functional crosstalk between mitochondria and extra-mitochondrial cellular environments plays a crucial role in the regulation of mitochondrial metabolism and physiology. Therefore, it is important to analyze mitochondria in vivo or in situ without their isolation from the natural cellular environment. This review summarizes previous studies and discusses existing approaches and methods for the analysis of mitochondrial function, structure, and intracellular organization in situ.

Dubious effects of methadone as an "anticancer" drug on ovarian cancer cell-lines and patient-derived tumor-spheroids.

BACKGROUND: The opioid agonist D,L-methadone exerts analgesic effects via the mu opioid receptor, encoded by OPRM1 and therefore plays a role in chronic pain management. In preclinical tumor-models D,L-methadone shows apoptotic and chemo-sensitizing effects and was therefore hyped as an off-label "anticancer" drug without substantiation from clinical trials. Its effects in ovarian cancer (OC) are completely unexplored. METHODS: We analyzed OPRM1-mRNA expression in six cisplatin-sensitive, two cisplatin-resistant OC cell-lines, 170 OC tissue samples and 12 non-neoplastic control tissues. Pro-angiogenetic, cytotoxic and apoptotic effects of D,L-methadone were evaluated in OC cell-lines and four patient-derived tumor-spheroid models. RESULTS: OPRM1 was transcriptionally expressed in 69% of OC-tissues and in three of eight OC cell-lines. D,L-methadone exposure significantly reduced cell-viability in five OC cell-lines irrespective of OPRM1 expression. D,L-methadone, applied alone or combined with cisplatin, showed no significant effects on apoptosis or VEGF secretion in cell-lines. Notably, in two of the four spheroid models, treatment with D,L-methadone significantly enhanced cell growth (by up to 121%), especially after long-term exposure. This is consistent with the observed attenuation of the inhibitory effects of cisplatin in three spheroid models when adding D,L-methadone. The effect of methadone treatment on VEGF secretion in tumor-spheroids was inconclusive. CONCLUSIONS: Our study demonstrates that certain OC samples express OPRM1, which, however, is not a prerequisite for D,L-methadone function. As such, D,L-methadone may exert also detrimental effects by stimulating the growth of certain OC-cells and abrogating cisplatin's therapeutic effect.

Lipidomic and Proteomic Alterations Induced by Even and Odd Medium-Chain Fatty Acids on Fibroblasts of Long-Chain Fatty Acid Oxidation Disorders.

Medium-chain fatty acids (mc-FAs) are currently applied in the treatment of long-chain fatty acid oxidation disorders (lc-FAOD) characterized by impaired ß-oxidation. Here, we performed lipidomic and proteomic analysis in fibroblasts from patients with very long-chain acyl-CoA dehydrogenase (VLCADD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHADD) deficiencies after incubation with heptanoate (C7) and octanoate (C8). Defects of ß-oxidation induced striking proteomic alterations, whereas the effect of treatment with mc-FAs was minor. However, mc-FAs induced a remodeling of complex lipids. Especially C7 appeared to act protectively by restoring sphingolipid biosynthesis flux and improving the observed dysregulation of protein homeostasis in LCHADD under control conditions.

3D bioprinting: novel approaches for engineering complex human tissue equivalents and drug testing.

Conventional approaches in drug development involve testing on 2D-cultured mammalian cells, followed by experiments in rodents. Although this is the common strategy, it has significant drawbacks: in 2D cell culture with human cells, the cultivation at normoxic conditions on a plastic or glass surface is an artificial situation that significantly changes energy metabolism, shape and intracellular signaling, which in turn directly affects drug response. On the other hand, rodents as the most frequently used animal models have evolutionarily separated from primates about 100 million years ago, with significant differences in physiology, which frequently leads to results not reproducible in humans. As an alternative, spheroid technology and micro-organoids have evolved in the last decade to provide 3D context for cells similar to native tissue. However, organoids used for drug testing are usually just in the 50-100 micrometers range and thereby too small to mimic micro-environmental tissue conditions such as limited nutrient and oxygen availability. An attractive alternative offers 3D bioprinting as this allows fabrication of human tissue equivalents from scratch with hollow structures for perfusion and strict spatiotemporal control over the deposition of cells and extracellular matrix proteins. Thereby, tissue surrogates with defined geometry are fabricated that offer unique opportunities in exploring cellular cross-talk, mechanobiology and morphogenesis. These tissue-equivalents are also very attractive tools in drug testing, as bioprinting enables standardized production, parallelization, and application-tailored design of human tissue, of human disease models and patient-specific tissue avatars. This review, therefore, summarizes recent advances in 3D bioprinting technology and its application for drug screening.

Different Lipid Signature in Fibroblasts of Long-Chain Fatty Acid Oxidation Disorders.

Long-chain fatty acid oxidation disorders (lc-FAOD) are a group of diseases affecting the degradation of long-chain fatty acids. In order to investigate the disease specific alterations of the cellular lipidome, we performed undirected lipidomics in fibroblasts from patients with carnitine palmitoyltransferase II, very long-chain acyl-CoA dehydrogenase, and long-chain 3-hydroxyacyl-CoA dehydrogenase. We demonstrate a deep remodeling of mitochondrial cardiolipins. The aberrant phosphatidylcholine/phosphatidylethanolamine ratio and the increased content of plasmalogens and of lysophospholipids support the theory of an inflammatory phenotype in lc-FAOD. Moreover, we describe increased ratios of sphingomyelin/ceramide and sphingomyelin/hexosylceramide in LCHAD deficiency which may contribute to the neuropathic phenotype of LCHADD/mitochondrial trifunctional protein deficiency.

Structural and functional remodeling of mitochondria as an adaptive response to energy deprivation.

Cancer cells bioenergetics is more dependent on glycolysis than mitochondrial oxidative phosphorylation, a phenomenon known as the Warburg Effect. It has been proposed that inhibition of glycolysis may selectively affect cancer cells. However, the effects of glycolysis inhibition on mitochondrial function and structure in cancer cells are not completely understood. Here, we investigated the comparative effects of 2-deoxy-d-glucose (2-DG, a glucose analogue, which suppresses cellular glycolysis) on cellular bioenergetics in human colon cancer DLD-1 cells, smooth muscle cells, human umbilical vein endothelial cells and HL-1 cardiomyocytes. In all cells, 2-DG treatment resulted in significant ATP depletion, however, the cell viability remained unchanged. Also, we did not observe the synergistic effects of 2-DG with anticancer drugs doxorubicin and 5-fluorouracil. Instead, after 2-DG treatment and ATP depletion, mitochondrial respiration and membrane potential were significantly enhanced and mitochondrial morphology changed in the direction of more network organization. Analysis of protein expression demonstrated that 2-DG treatment induced an activation of AMPK (elevated pAMPK/AMPK ratio), increased mitochondrial fusion (mitofusins 1 and 2) and decreased fission (Drp1) proteins. In conclusion, this study suggests a strong link between respiratory function and structural organization of mitochondria in the cell. We propose that the functionality of the mitochondrial network is enhanced compared to disconnected mitochondria.

Crosstalk between Mitochondria and Cytoskeleton in Cardiac Cells.

Elucidation of the mitochondrial regulatory mechanisms for the understanding of muscle bioenergetics and the role of mitochondria is a fundamental problem in cellular physiology and pathophysiology. The cytoskeleton (microtubules, intermediate filaments, microfilaments) plays a central role in the maintenance of mitochondrial shape, location, and motility. In addition, numerous interactions between cytoskeletal proteins and mitochondria can actively participate in the regulation of mitochondrial respiration and oxidative phosphorylation. In cardiac and skeletal muscles, mitochondrial positions are tightly fixed, providing their regular arrangement and numerous interactions with other cellular structures such as sarcoplasmic reticulum and cytoskeleton. This can involve association of cytoskeletal proteins with voltage-dependent anion channel (VDAC), thereby, governing the permeability of the outer mitochondrial membrane (OMM) to metabolites, and regulating cell energy metabolism. Cardiomyocytes and myocardial fibers demonstrate regular arrangement of tubulin beta-II isoform entirely co-localized with mitochondria, in contrast to other isoforms of tubulin. This observation suggests the participation of tubulin beta-II in the regulation of OMM permeability through interaction with VDAC. The OMM permeability is also regulated by the specific isoform of cytolinker protein plectin. This review summarizes and discusses previous studies on the role of cytoskeletal proteins in the regulation of energy metabolism and mitochondrial function, adenosine triphosphate (ATP) production, and energy transfer.

A drug library screen identifies Carbenoxolone as novel FOXO inhibitor that overcomes FOXO3-mediated chemoprotection in high-stage neuroblastoma.

The transcription factor FOXO3 has been associated in different tumor entities with hallmarks of cancer, including metastasis, tumor angiogenesis, maintenance of tumor-initiating stem cells, and drug resistance. In neuroblastoma (NB), we recently demonstrated that nuclear FOXO3 promotes tumor angiogenesis in vivo and chemoresistance in vitro. Hence, inhibiting the transcriptional activity of FOXO3 is a promising therapeutic strategy. However, as no FOXO3 inhibitor is clinically available to date, we used a medium-throughput fluorescence polarization assay (FPA) screening in a drug-repositioning approach to identify compounds that bind to the FOXO3-DNA-binding-domain (DBD). Carbenoxolone (CBX), a glycyrrhetinic acid derivative, was identified as a potential FOXO3-inhibitory compound that binds to the FOXO3-DBD with a binding affinity of 19 µM. Specific interaction of CBX with the FOXO3-DBD was validated by fluorescence-based electrophoretic mobility shift assay (FAM-EMSA). CBX inhibits the transcriptional activity of FOXO3 target genes, as determined by chromatin immunoprecipitation (ChIP), DEPP-, and BIM promoter reporter assays, and real-time RT-PCR analyses. In high-stage NB cells with functional TP53, FOXO3 triggers the expression of SESN3, which increases chemoprotection and cell survival. Importantly, FOXO3 inhibition by CBX treatment at pharmacologically relevant concentrations efficiently repressed FOXO3-mediated SESN3 expression and clonogenic survival and sensitized high-stage NB cells to chemotherapy in a 2D and 3D culture model. Thus, CBX might be a promising novel candidate for the treatment of therapy-resistant high-stage NB and other "FOXO-resistant" cancers.

Repaglinide Silences the FOXO3/Lumican Axis and Represses the Associated Metastatic Potential of Neuronal Cancer Cells.

The transcription factor FOXO3 is associated with poor outcome in high-stage neuroblastoma (NB), as it facilitates chemoprotection and tumor angiogenesis. In other tumor entities, FOXO3 stimulates metastasis formation, one of the biggest challenges in the treatment of aggressive NB. However, the impact of FOXO3 on the metastatic potential of neuronal tumor cells remains largely unknown. In the present study, we uncover the small leucine-rich proteoglycan family member lumican (LUM) as a FOXO3-regulated gene that stimulates cellular migration in NB. By a drug-library screen we identified the small molecular weight compound repaglinide (RPG) as a putative FOXO3 inhibitor. Here, we verify that RPG binds to the FOXO3-DNA-binding-domain (DBD) and thereby silences the transcriptional activity of FOXO3. Consistent with the concept that the FOXO3/LUM axis enhances the migratory capacity of aggressive NB cells, we demonstrate that stable knockdown of LUM abrogates the FOXO3-mediated increase in cellular migration. Importantly, FOXO3 inhibition by RPG represses the binding of FOXO3 to the LUM promoter, inhibits FOXO3-mediated LUM RNA and protein expression, and efficiently abrogates FOXO3-triggered cellular "wound healing" as well as spheroid-based 3D-migration. Thus, silencing the FOXO3/LUM axis by the FDA-approved compound RPG represents a promising strategy for novel therapeutic interventions in NB and other FOXO3-dependent tumors.

Modulating FOXO3 transcriptional activity by small, DBD-binding molecules.

FOXO transcription factors are critical regulators of cell homeostasis and steer cell death, differentiation and longevity in mammalian cells. By combined pharmacophore-modeling-based in silico and fluorescence polarization-based screening we identified small molecules that physically interact with the DNA-binding domain (DBD) of FOXO3 and modulate the FOXO3 transcriptional program in human cells. The mode of interaction between compounds and the FOXO3-DBD was assessed via NMR spectroscopy and docking studies. We demonstrate that compounds S9 and its oxalate salt S9OX interfere with FOXO3 target promoter binding, gene transcription and modulate the physiologic program activated by FOXO3 in cancer cells. These small molecules prove the druggability of the FOXO-DBD and provide a structural basis for modulating these important homeostasis regulators in normal and malignant cells.

The Role of Mitochondria in the Mechanisms of Cardiac Ischemia-Reperfusion Injury.

Mitochondria play a critical role in maintaining cellular function by ATP production. They are also a source of reactive oxygen species (ROS) and proapoptotic factors. The role of mitochondria has been established in many aspects of cell physiology/pathophysiology, including cell signaling. Mitochondria may deteriorate under various pathological conditions, including ischemia-reperfusion (IR) injury. Mitochondrial injury can be one of the main causes for cardiac and other tissue injuries by energy stress and overproduction of toxic reactive oxygen species, leading to oxidative stress, elevated calcium and apoptotic and necrotic cell death. However, the interplay among these processes in normal and pathological conditions is still poorly understood. Mitochondria play a critical role in cardiac IR injury, where they are directly involved in several pathophysiological mechanisms. We also discuss the role of mitochondria in the context of mitochondrial dynamics, specializations and heterogeneity. Also, we wanted to stress the existence of morphologically and functionally different mitochondrial subpopulations in the heart that may have different sensitivities to diseases and IR injury. Therefore, various cardioprotective interventions that modulate mitochondrial stability, dynamics and turnover, including various pharmacologic agents, specific mitochondrial antioxidants and uncouplers, and ischemic preconditioning can be considered as the main strategies to protect mitochondrial and cardiovascular function and thus enhance longevity.

Modulation of Respiration and Mitochondrial Dynamics by SMAC-Mimetics for Combination Therapy in Chemoresistant Cancer.

Inhibitor of apoptosis proteins (IAP) are cell death regulators that bind caspases and interfere with apoptotic signalling via death receptors or intrinsic cell death pathways. BIRC4/XIAP is the most potent anti-apoptotic IAP-member and it physically interacts with caspases via its BIR2 and its BIR3 domain. These domains are also critical for the interaction with mitochondria-derived SMAC/Diablo and with the IAP protein survivin. Survivin is frequently overexpressed in neuroblastoma due to a gain of 17q and we have demonstrated that survivin confers resistance to chemotherapeutic agents and reprograms metabolism of neuroblastoma cells towards glycolysis. As regulator of mitochondrial fission and autophagy survivin acts at the crossroads of mitochondrial architecture, autophagy and cellular energy metabolism. Methods: We tested the effect of SMAC-mimetics on the XIAP/survivin axis as modulator of cellular metabolism analysing mitochondrial morphology, metabolic intermediates and cellular survival. Finally, the impact of the combined treatment was evaluated in a xenograft neuroblastoma mouse model assessing the therapy effect on tumour size and volume. Results: Here we demonstrated that XIAP sequesters significant amounts of survivin within the cell that can be mobilized by so called SMAC-mimetics. SMAC-mimetics are drugs that are designed to bind with high affinity to XIAP-BIR2 / BIR3 domains to release caspases and re-sensitize XIAP-overexpressing tumors for chemotherapy. However, SMAC-mimetic treatment releases also survivin from XIAP and thereby induces mitochondrial fragmentation, prevents ROS accumulation and leads to the Warburg effect, an unwanted side effect of this therapy. Importantly, cells that drift into a highly glycolytic state due to SMAC-mimetic treatment become also highly sensitive to non-genotoxic treatment with glycolysis inhibitors such as 2-Deoxy-D-glucose (2DG) in vitro and in vivo. Conclusion: A combinational therapy of non-genotoxic SMAC-mimetics and glycolysis-inhibitors overcomes IAP-mediated cell survival in cancer and provides therefore an attractive usage of SMAC-mimetics.

Forkhead Domains of FOXO Transcription Factors Differ in both Overall Conformation and Dynamics.

FOXO transcription factors regulate cellular homeostasis, longevity and response to stress. FOXO1 (also known as FKHR) is a key regulator of hepatic glucose production and lipid metabolism, and its specific inhibition may have beneficial effects on diabetic hyperglycemia by reducing hepatic glucose production. Moreover, all FOXO proteins are considered potential drug targets for drug resistance prevention in cancer therapy. However, the development of specific FOXO inhibitors requires a detailed understanding of structural differences between individual FOXO DNA-binding domains. The high-resolution structure of the DNA-binding domain of FOXO1 reported in this study and its comparison with structures of other FOXO proteins revealed differences in both their conformation and flexibility. These differences are encoded by variations in protein sequences and account for the distinct functions of FOXO proteins. In particular, the positions of the helices H1, H2 and H3, whose interface form the hydrophobic core of the Forkhead domain, and the interactions between hydrophobic residues located on the interface between the N-terminal segment, the H2-H3 loop, and the recognition helix H3 differ among apo FOXO1, FOXO3 and FOXO4 proteins. Therefore, the availability of apo structures of DNA-binding domains of all three major FOXO proteins will support the development of FOXO-type-specific inhibitors.

Very long-/ and long Chain-3-Hydroxy Acyl CoA Dehydrogenase Deficiency correlates with deregulation of the mitochondrial fusion/fission machinery.

Children diagnosed with Long-Chain-3-Hydroxy-Acyl-CoA-Dehydrogenase-Deficiency (LCHADD) or Very-Long-Chain-3-Hydroxy-Acyl-CoA-Dehydrogenase-Deficiency (VLCADD) frequently present with hypertrophic cardiomyopathy or muscle weakness which is caused by the accumulation of fatty acid metabolites due to inactivating mutations in the mitochondrial trifunctional protein. By analyzing mitochondrial morphology we uncovered that mutations within the HADHA or the ACADVL gene not only affect fatty acid oxidation, but also cause significant changes in the DNM1L/MFN2 ratio leading to the significant accumulation of truncated and punctate mitochondria in contrast to network-like mitochondrial morphology in controls. These striking morphological abnormalities correlate with changes in OXPHOS, an imbalance in ROS levels, reduced mitochondrial respiration, reduced growth rates and significantly increased glucose uptake per cell, suggesting that HADHA and ACADVL mutations shift cellular energy household into glycolysis. Experiments using the NOX2-specific inhibitor Phox-I2 suggest that NOX2 is activated by accumulating long-chain fatty acids and generates ROS, which in turn changes mitochondrial morphology and activity. We thereby provide novel insights into the cellular energy household of cells from LCHADD/VLCADD patients and demonstrate for the first time a connection between fatty acid metabolism, mitochondrial morphology and ROS in patients with these rare genetic disorders.

The tubulin inhibitor MG-2477 induces autophagy-regulated cell death, ROS accumulation and activation of FOXO3 in neuroblastoma.

Neuroblastoma is the most frequent extra-cranial solid tumor in children with still high mortality in stage M. Here we studied the tubulin-inhibitor MG-2477 as a possible therapeutic agent for neuroblastoma therapy and uncovered that MG-2477 induces death in neuroblastoma cells independent of PKB-activation status and stage. MG-2477 triggers within 30 minutes extensive autophagosome-formation that finally leads to cell death associated with mitotic catastrophe. Autophagy is critical for MG-2477-induced death and is regulated by the BH3-only protein PMAIP1/NOXA which sequesters the anti-apoptotic BCL2-protein BCLXL and thereby displaces and activates the autophagy-regulator BECN1/beclin1. Knockdown of NOXA or overexpression of its pro-survival binding partners MCL1 and BCLXL counteracts MG-2477-induced cell death. MG-2477 also rapidly induces the repression of the anti-apoptotic protein Survivin, which promotes autophagy and cell death. We further observed the accumulation of reactive oxygen species (ROS) that triggers autophagy induction suggesting a change of the PI3 kinase-III/BECN1 complex and activates the transcription factor FOXO3, which contributes to final cell death induction. The combined data suggest that MG-2477 induces a sequential process of ROS-accumulation, autophagy and FOXO3-activation that leads to cell death in neuroblastoma cells.

Nuclear FOXO3 predicts adverse clinical outcome and promotes tumor angiogenesis in neuroblastoma.

Neuroblastoma is the most frequent, extracranial solid tumor in children with still poor prognosis in stage IV disease. In this study, we analyzed FOXO3-phosphorylation and cellular localization in tumor biopsies and determined the function of this homeostasis regulator in vitro and in vivo. FOXO3-phosphorylation at threonine-32 (T32) and nuclear localization in biopsies significantly correlated with stage IV disease. DNA-damaging drugs induced nuclear accumulation of FOXO3, which was associated with elevated T32-phosphorylation in stage IV-derived neuroblastoma cells, thereby reflecting the in situ results. In contrast, hypoxic conditions repressed PKB-activity and caused dephosphorylation of FOXO3 in both, stroma-like SH-EP and high-stage-derived STA-NB15 cells. The activation of an ectopically-expressed FOXO3 in these cells reduced viability at normoxia, but promoted growth at hypoxic conditions and elevated VEGF-C-expression. In chorioallantoic membrane (CAM) assays STA-NB15 tumors with ectopic FOXO3 showed increased micro-vessel formation and, when xenografted into nude mice, a gene-dosage-dependent effect of FOXO3 in high-stage STA-NB15 cells became evident: low-level activation increased tumor-vascularization, whereas hyper-activation repressed tumor growth.The combined data suggest that, depending on the mode and intensity of activation, cellular FOXO3 acts as a homeostasis regulator promoting tumor growth at hypoxic conditions and tumor angiogenesis in high-stage neuroblastoma.