Neuroblastoma is the most common solid tumor in childhood and develops from undifferentiated progenitor cells of the sympathetic nervous system. In neuronal tumor cells DNA-damaging chemotherapeutic agents activate the transcription factor FOXO3 which regulates the formation of reactive oxygen species (ROS) and cell death as well as a longevity program associated with therapy resistance. We demonstrated before that C10ORF10/DEPP, a transcriptional target of FOXO3, localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification. In the present study, we investigated the impact of FOXO3 and DEPP on the regulation of autophagy. Autophagy serves to reduce oxidative damage as it triggers a self-degradative process for the removal of aggregated or misfolded proteins and damaged organelles. The effect of FOXO3 and DEPP on autophagy induction was analyzed using live cell fluorescence microscopy and immunoblot analyses of SH-EP cells transfected with a plasmid for EYFP-LC3 and with siRNAs specific for LC3, respectively. ROS steady-state levels were measured with reduced MitoTrackerRed CM-H2XROS. Cellular apoptosis was analyzed by flow cytometry and the caspase 3/7 assay. We report for the first time that DEPP induces ROS accumulation and thereby mediates the formation of autophagosomes as inhibition of ROS formation by N-acetyl-cysteine completely blocks autophagy. We further demonstrate that H2O2-treatment triggers autophagy-induction by FOXO3-mediated DEPP expression. Importantly, knockdown of DEPP was sufficient to efficiently inhibit autophagy-induction under different stress conditions such as serum starvation and genotoxic stress, suggesting that DEPP expression is critical for the initiation of autophagy in neuroblastoma. FOXO3-triggered autophagy partially protects neuroblastoma cells from cell death. Consistent with this concept, we demonstrate that inhibition of autophagy by LC3-knockdown significantly increased etoposide- and doxorubicin-induced apoptosis. These results were also confirmed by the use of the autophagy-inhibitor chloroquine that significantly enhanced the chemotherapeutic effect of etoposide and doxorubicin in neuronal tumor cells. Targeting FOXO3/DEPP-triggered autophagy is a promising strategy to sensitize neuroblastoma cells to chemotherapy.

Normothermic machine perfusion (NMP) has emerged as an innovative organ preservation technique. Developing an understanding for the donor organ immune cell composition and its dynamic changes during NMP is essential. We aimed for a comprehensive characterization of immune cell (sub)populations, cell trafficking and cytokine release during liver NMP. Single-cell transcriptome profiling of human donor livers prior to, during NMP and after transplantation shows an abundance of CXC chemokine receptor 1⁺/2⁺ (CXCR1⁺/CXCR2⁺) neutrophils, which significantly decreased during NMP. This is paralleled by a large efflux of passenger leukocytes with neutrophil predominance in the perfusate. During NMP, neutrophils shift from a pro-inflammatory state towards an aged/chronically activated/exhausted phenotype, while anti-inflammatory/tolerogenic monocytes/macrophages are increased. We herein describe the dynamics of the immune cell repertoire, phenotypic immune cell shifts and a dominance of neutrophils during liver NMP, which potentially contribute to the inflammatory response. Our findings may serve as resource to initiate future immune-interventional studies.

FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells. The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor. We report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells. The combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.

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.

The development of single-cell omics tools has enabled scientists to study the tumor microenvironment (TME) in unprecedented detail. However, each of the different techniques may have its unique strengths and limitations. Here we directly compared two commercially available high-throughput single-cell RNA sequencing (scRNA-seq) technologies - droplet-based 10X Chromium vs. microwell-based BD Rhapsody - using paired samples from patients with localized prostate cancer (PCa) undergoing a radical prostatectomy.