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Interferons (IFNs) are key players in the tumor immune response and act by inducing the expression of IFN-stimulated genes (ISGs). Here, we identify the mixed-lineage kinase domain-like pseudokinase (MLKL) as an ISG in various cancer cell lines. Both type I and type II IFNs increase the expression of MLKL indicating that MLKL up-regulation is a general feature of IFN signaling. IFNγ up-regulates mRNA as well as protein levels of MLKL demonstrating that IFNγ transcriptionally regulates MLKL. This notion is further supported by Actinomycin D chase experiments showing that IFNγ-stimulated up-regulation of MLKL is prevented in the presence of the transcriptional inhibitor Actinomycin D. Also, knockdown of the transcription factor IFN-regulatory factor 1 (IRF1) and signal transducer and activator of transcription (STAT) 1 as well as knockout of IRF1 significantly attenuate IFNγ-mediated induction of MLKL mRNA levels. Up-regulation of MLKL by IFNγ provides a valuable tool to sensitize cells towards necroptotic cell death and to overcome apoptosis resistance of cancer cells.
Loperamide, pimozide, and STF-62247 trigger autophagy-dependent cell death in glioblastoma cells
(2018)
Autophagy is a well-described degradation mechanism that promotes cell survival upon nutrient starvation and other forms of cellular stresses. In addition, there is growing evidence showing that autophagy can exert a lethal function via autophagic cell death (ACD). As ACD has been implicated in apoptosis-resistant glioblastoma (GBM), there is a high medical need for identifying novel ACD-inducing drugs. Therefore, we screened a library containing 70 autophagy-inducing compounds to induce ATG5-dependent cell death in human MZ-54 GBM cells. Here, we identified three compounds, i.e. loperamide, pimozide, and STF-62247 that significantly induce cell death in several GBM cell lines compared to CRISPR/Cas9-generated ATG5- or ATG7-deficient cells, pointing to a death-promoting role of autophagy. Further cell death analyses conducted using pharmacological inhibitors revealed that apoptosis, ferroptosis, and necroptosis only play minor roles in loperamide-, pimozide- or STF-62247-induced cell death. Intriguingly, these three compounds induce massive lipidation of the autophagy marker protein LC3B as well as the formation of LC3B puncta, which are characteristic of autophagy. Furthermore, loperamide, pimozide, and STF-62247 enhance the autophagic flux in parental MZ-54 cells, but not in ATG5 or ATG7 knockout (KO) MZ-54 cells. In addition, loperamide- and pimozide-treated cells display a massive formation of autophagosomes and autolysosomes at the ultrastructural level. Finally, stimulation of autophagy by all three compounds is accompanied by dephosphorylation of mammalian target of rapamycin complex 1 (mTORC1), a well-known negative regulator of autophagy. In summary, our results indicate that loperamide, pimozide, and STF-62247 induce ATG5- and ATG7-dependent cell death in GBM cells, which is preceded by a massive induction of autophagy. These findings emphasize the lethal function and potential clinical relevance of hyperactivated autophagy in GBM.
Background: Altered neuronal development is discussed as the underlying pathogenic mechanism of autism spectrum disorders (ASD). Copy number variations of 16p11.2 have recurrently been identified in individuals with ASD. Of the 29 genes within this region, quinolinate phosphoribosyltransferase (QPRT) showed the strongest regulation during neuronal differentiation of SH-SY5Y neuroblastoma cells. We hypothesized a causal relation between this tryptophan metabolism-related enzyme and neuronal differentiation. We thus analyzed the effect of QPRT on the differentiation of SH-SY5Y and specifically focused on neuronal morphology, metabolites of the tryptophan pathway, and the neurodevelopmental transcriptome.
Methods: The gene dosage-dependent change of QPRT expression following Chr16p11.2 deletion was investigated in a lymphoblastoid cell line (LCL) of a deletion carrier and compared to his non-carrier parents. Expression of QPRT was tested for correlation with neuromorphology in SH-SY5Y cells. QPRT function was inhibited in SH-SY5Y neuroblastoma cells using (i) siRNA knockdown (KD), (ii) chemical mimicking of loss of QPRT, and (iii) complete CRISPR/Cas9-mediated knock out (KO). QPRT-KD cells underwent morphological analysis. Chemically inhibited and QPRT-KO cells were characterized using viability assays. Additionally, QPRT-KO cells underwent metabolite and whole transcriptome analyses. Genes differentially expressed upon KO of QPRT were tested for enrichment in biological processes and co-regulated gene-networks of the human brain.
Results: QPRT expression was reduced in the LCL of the deletion carrier and significantly correlated with the neuritic complexity of SH-SY5Y. The reduction of QPRT altered neuronal morphology of differentiated SH-SY5Y cells. Chemical inhibition as well as complete KO of the gene were lethal upon induction of neuronal differentiation, but not proliferation. The QPRT-associated tryptophan pathway was not affected by KO. At the transcriptome level, genes linked to neurodevelopmental processes and synaptic structures were affected. Differentially regulated genes were enriched for ASD candidates, and co-regulated gene networks were implicated in the development of the dorsolateral prefrontal cortex, the hippocampus, and the amygdala.
Conclusions: In this study, QPRT was causally related to in vitro neuronal differentiation of SH-SY5Y cells and affected the regulation of genes and gene networks previously implicated in ASD. Thus, our data suggest that QPRT may play an important role in the pathogenesis of ASD in Chr16p11.2 deletion carriers.
Background: Signal transduction pathways are important cellular processes to maintain the cell’s integrity. Their imbalance can cause severe pathologies. As signal transduction pathways feature complex regulations, they form intertwined networks. Mathematical models aim to capture their regulatory logic and allow an unbiased analysis of robustness and vulnerability of the signaling network. Pathway detection is yet a challenge for the analysis of signaling networks in the field of systems biology. A rigorous mathematical formalism is lacking to identify all possible signal flows in a network model.
Results: In this paper, we introduce the concept of Manatee invariants for the analysis of signal transduction networks. We present an algorithm for the characterization of the combinatorial diversity of signal flows, e.g., from signal reception to cellular response. We demonstrate the concept for a small model of the TNFR1-mediated NF- κB signaling pathway. Manatee invariants reveal all possible signal flows in the network. Further, we show the application of Manatee invariants for in silico knockout experiments. Here, we illustrate the biological relevance of the concept.
Conclusions: The proposed mathematical framework reveals the entire variety of signal flows in models of signaling systems, including cyclic regulations. Thereby, Manatee invariants allow for the analysis of robustness and vulnerability of signaling networks. The application to further analyses such as for in silico knockout was shown. The new framework of Manatee invariants contributes to an advanced examination of signaling systems.
Mathematical modeling of the molecular switch of TNFR1-mediated signaling pathways using Petri nets
(2021)
The paper describes a mathematical model of the molecular switch of cell survival, apoptosis, and necroptosis in cellular signaling pathways initiated by tumor necrosis factor 1. Based on experimental findings in the current literature, we constructed a Petri net model in terms of detailed molecular reactions for the molecular players, protein complexes, post-translational modifications, and cross talk. The model comprises 118 biochemical entities, 130 reactions, and 299 connecting edges. Applying Petri net analysis techniques, we found 279 pathways describing complete signal flows from receptor activation to cellular response, representing the combinatorial diversity of functional pathways.120 pathways steered the cell to survival, whereas 58 and 35 pathways led to apoptosis and necroptosis, respectively. For 65 pathways, the triggered response was not deterministic, leading to multiple possible outcomes. Based on the Petri net, we investigated the detailed in silico knockout behavior and identified important checkpoints of the TNFR1 signaling pathway in terms of ubiquitination within complex I and the gene expression dependent on NF-κB, which controls the caspase activity in complex II and apoptosis induction.
Background: NH exchangers (NHEs) play a crucial role in regulating intra/extracellular pH, which is altered in cancer cells, and are therefore suitable targets to alter cancer cell metabolism in order to inhibit cell survival and proliferation. Among NHE inhibitors, amiloride family members are commonly used in clinical practice as diuretics; we focused on the amiloride HMA, reporting a net cytotoxic effect on a panel of human cancer cell lines; now we aim to provide new insights into the molecular events leading to cell death by HMA.
Methods: Colon cancer cell lines were treated with HMA and analysed with: morphological and cellular assays for cell viability and death, and autophagy; biochemical approaches to evaluate mitochondrial function and ROS production; in situ detection of DNA damage; molecular tools to silence crucial autophagy/necroptosis factors.
Results: HMA affects cellular morphology, alters mitochondrial structure and function, causes an increase in ROS, which is detrimental to DNA integrity, stimulates poly(ADP-ribose) synthesis, activates RIPK3-dependent death and triggers autophagy, which is unable to rescue cell survival. These features are hot points of an intricate network of processes, including necroptosis and autophagy, regulating the homeostasis between survival and death.
Conclusion: Our results allow the identification of multiple events leading to cell death in cancer cells treated with HMA. The here-defined intricate network activated by HMA could be instrumental to selectively target the key players of each pathway in the attempt to improve the global response to HMA. Our data could be the starting point for developing a newly designed targeted therapy.
Lysine-specific demethylase 1 (LSD1), a histone lysine demethylase with the main specificity for H3K4me2, has been shown to be overexpressed in rhabdomyosarcoma (RMS) tumor samples. However, its role in RMS biology is not yet well understood. Here, we identified a new role of LSD1 in regulating adhesion of RMS cells. Genetic knockdown of LSD1 profoundly suppressed clonogenic growth in a panel of RMS cell lines, whereas LSD1 proved to be largely dispensable for regulating cell death and short-term survival. Combined RNA and ChIP-sequencing performed to analyze RNA expression and histone methylation at promoter regions revealed a gene set enrichment for adhesion-associated terms upon LSD1 knockdown. Consistently, LSD1 knockdown significantly reduced adhesion to untreated surfaces. Importantly, precoating of the plates with the adhesives collagen I or fibronectin rescued this reduced adhesion of LSD1 knockdown cells back to levels of control cells. Using KEGG pathway analysis, we identified 17 differentially expressed genes (DEGs) in LSD1 knockdown cells related to adhesion processes, which were validated by qRT-PCR. Combining RNA and ChIP-sequencing results revealed that, within this set of genes, SPP1, C3AR1, ITGA10 and SERPINE1 also exhibited increased H3K4me2 levels at their promoter regions in LSD1 knockdown compared to control cells. Indeed, LSD1 ChIP experiments confirmed enrichment of LSD1 at their promoter regions, suggesting a direct transcriptional regulation by LSD1. By identifying a new role of LSD1 in the modulation of cell adhesion and clonogenic growth of RMS cells, these findings highlight the importance of LSD1 in RMS.
NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation
Novel insights into the synergistic interaction of Bortezomib and TRAIL: tBid provides the link
(2011)
The proteasome inhibitor Bortezomib has been identified as a potent enhancer of TRAIL-induced apoptosis in several human cancers. However, the identification of the underlying molecular mechanisms of this synergistic cell death induction has been ongoing over the last years. A recent study identifies a new mechanism of action for the synergism of TRAIL and Bortezomib.
During polychemotherapy, cytotoxic drugs are given in combinations to enhance their anti-tumor effectiveness. For most drug combinations, underlying signaling mechanisms responsible for positive drug-drug interactions remain elusive. Here, we prove a decisive role for the Bcl-2 family member NOXA to mediate cell death by certain drug combinations, even if drugs were combined which acted independently from NOXA, when given alone. In proof-of-principle studies, betulinic acid, doxorubicin and vincristine induced cell death in a p53- and NOXA-independent pathway involving mitochondrial pore formation, release of cytochrome c and caspase activation. In contrast, when betulinic acid was combined with either doxorubicine or vincristine, cell death signaling changed considerably; the drug combinations clearly depended on both p53 and NOXA. Similarly and of high clinical relevance, in patient-derived childhood acute leukemia samples the drug combinations, but not the single drugs depended on p53 and NOXA, as shown by RNA interference studies in patient-derived cells. Our data emphasize that NOXA represents an important target molecule for combinations of drugs that alone do not target NOXA. NOXA might have a special role in regulating apoptosis sensitivity in the complex interplay of polychemotherapy. Deciphering the differences in signaling of single drugs and drug combinations might enable designing highly effective novel polychemotherapy regimens.