Stress-induced TRAILR2 expression overcomes TRAIL resistance in cancer cell spheroids

The influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated

Genetic variation in insulin-like growth factor signaling genes and breast cancer risk among BRCA1 and BRCA2 carriers

Abstract Introduction Women who carry mutations in BRCA1 and BRCA2 have a substantially increased risk of developing breast cancer as compared with the general population. However, risk estimates range from 20 to 80%, suggesting the presence of genetic and/or environmental risk modifiers. Based on extensive in vivo and in vitro studies, one important pathway for breast cancer pathogenesis may be the insulin-like growth factor (IGF) signaling pathway, which regulates both cellular proliferation and apoptosis. BRCA1 has been shown to directly interact with IGF signaling such that variants in this pathway may modify risk of cancer in women carrying BRCA mutations. In this study, we investigate the association of variants in genes involved in IGF signaling and risk of breast cancer in women who carry deleterious BRCA1 and BRCA2 mutations. Methods A cohort of 1,665 adult, female mutation carriers, including 1,122 BRCA1 carriers (433 cases) and 543 BRCA2 carriers (238 cases) were genotyped for SNPs in IGF1, IGF1 receptor (IGF1R), IGF1 binding protein (IGFBP1, IGFBP2, IGFBP5), and IGF receptor substrate 1 (IRS1). Cox proportional hazards regression was used to model time from birth to diagnosis of breast cancer for BRCA1 and BRCA2 carriers separately. For linkage disequilibrium (LD) blocks with multiple SNPs, an additive genetic model was assumed; and for single SNP analyses, no additivity assumptions were made. Results Among BRCA1 carriers, significant associations were found between risk of breast cancer and LD blocks in IGF1R (global P = 0.011 for LD block 2 and global P = 0.012 for LD block 11). Among BRCA2 carriers, an LD block in IGFBP2 (global P = 0.0145) was found to be associated with the time to breast cancer diagnosis. No significant LD block associations were found for the other investigated genes among BRCA1 and BRCA2 carriers. Conclusions This is the first study to investigate the role of genetic variation in IGF signaling and breast cancer risk in women carrying deleterious mutations in BRCA1 and BRCA2. We identified significant associations in variants in IGF1R and IRS1 in BRCA1 carriers and in IGFBP2 in BRCA2 carriers. Although there is known to be interaction of BRCA1 and IGF signaling, further replication and identification of causal mechanisms are needed to better understand these associations

Para-infectious brain injury in COVID-19 persists at follow-up despite attenuated cytokine and autoantibody responses

To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1–11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury. Autoantibodies are more common in COVID-19 than controls and some (including against MYL7, UCH-L1, and GRIN3B) are more frequent with altered consciousness. Additionally, convalescent participants with neurological complications show elevated GFAP and NfL, unrelated to attenuated systemic inflammatory mediators and to autoantibody responses. Overall, neurological complications of COVID-19 are associated with evidence of neuroglial injury in both acute and late disease and these correlate with dysregulated innate and adaptive immune responses acutely

Functions and Regulation of eukaryotic NAD kinase - an essential enzyme for NADP biosynthesis

NAD and NADP, both major carriers of cellular reducing equivalents, are of vital importance in diverse metabolic and signaling processes. Efficient maintenance and regulation of the reduction-oxidation status is therefore crucial within the cell. The only known pathway of NADP generation is the phosphorylation of NAD catalyzed by NAD kinase (NADK). Owing to the multiple roles of NADP these enzymes have gained considerable interest over the past few years. Genes encoding NADKs have been cloned only recently. The enzymatic activity has been shown to be essential for prokaryotic organisms and important for maintenance of the NADP pool in yeast Saccharomyces cerevisiae and plant Arabidopsis thaliana. While both yeast and plant encode three isoforms, only a single NADK, located in the cytoplasm, has been identified in mammals. Little is known about the function and regulation of NADK in mammalian cells. To investigate the physiological roles of NADK its expression level was altered in human cells. While recombinant NADK was rather specific for the oxidized form, NAD+, as substrate, modulation of cellular NADK activity was reflected in significant changes of the NADPH content. Overexpression of NADK caused a 4-5 fold increase in NADPH but provided only moderate protection towards oxidative stress conditions. Shorthairpin- RNA-mediated downregulation of NADK had surprisingly little impact on cell proliferation, endogenous reactive oxygen species levels and cell viability after oxidant treatment. However, the stable shifts in the NADP redox ratio in the generated cell lines influenced the expression of genes known to be involved in oxidative stress response. Furthermore, phosphorylation of NADK in vitro mediated by calcium/calmodulin-dependent protein kinase II was established. The serine residue 64 was identified as phosphorylation site. The consequence of this modification remains unclear since no influence on the catalytic activity or the subcellular localization of NADK was observed. Two NADK isoforms, which differ only in their N-terminal sequences, were identified in the sea urchin Strongylocentrotus purpuratus. Importantly, for both isoforms a direct activation of NADK by calcium/calmodulin was demonstrated. Although the existence of calmodulindependent NADKs has long been appreciated, the molecular basis of this regulation had so far remained unknown. Moreover, one of the isoforms was also specifically phosphorylated by calcium/calmodulin-dependent protein kinase II. The calmodulin-mediated activation and phosphorylation was associated with the different N-termini, while the catalytic domain is found in the identical C-terminal region of the proteins. The results suggest the N-terminal part of NADKs to be important as a regulatory domain

Sample-based modeling reveals bidirectional interplay between cell cycle progression and extrinsic apoptosis.

Apoptotic cell death can be initiated through the extrinsic and intrinsic signaling pathways. While cell cycle progression promotes the responsiveness to intrinsic apoptosis induced by genotoxic stress or spindle poisons, this has not yet been studied conclusively for extrinsic apoptosis. Here, we combined fluorescence-based time-lapse monitoring of cell cycle progression and cell death execution by long-term time-lapse microscopy with sampling-based mathematical modeling to study cell cycle dependency of TRAIL-induced extrinsic apoptosis in NCI-H460/geminin cells. In particular, we investigated the interaction of cell death timing and progression of cell cycle states. We not only found that TRAIL prolongs cycle progression, but in reverse also that cell cycle progression affects the kinetics of TRAIL-induced apoptosis: Cells exposed to TRAIL in G1 died significantly faster than cells stimulated in S/G2/M. The connection between cell cycle state and apoptosis progression was captured by developing a mathematical model, for which parameter estimation revealed that apoptosis progression decelerates in the second half of the cell cycle. Similar results were also obtained when studying HCT-116 cells. Our results therefore reject the null hypothesis of independence between cell cycle progression and extrinsic apoptosis and, supported by simulations and experiments of synchronized cell populations, suggest that unwanted escape from TRAIL-induced apoptosis can be reduced by enriching the fraction of cells in G1 phase. Besides novel insight into the interrelation of cell cycle progression and extrinsic apoptosis signaling kinetics, our findings are therefore also relevant for optimizing future TRAIL-based treatment strategies

Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps.

Dysregulation of the mitochondrial signaling pathway of apoptosis induction represents a major hurdle in tumor therapy. The objective of the presented work was to investigate the role of the intrinsic (mitochondrial) apoptotic pathway in the non-small lung cancer cell line NCI-H460 upon induction of apoptosis using the highly bioactive TRAIL derivative Db-scTRAIL. NCI-H460 cells were TRAIL sensitive but an only about 3 fold overexpression of Bcl-2 was sufficient to induce a highly TRAIL resistant phenotype, confirming that the mitochondrial pathway is crucial for TRAIL-induced apoptosis induction. TRAIL resistance was paralleled by a strong inhibition of caspase-8, -9 and -3 activities and blocked their full processing. Notably, especially the final cleavage steps of the initiator caspase-8 and the executioner caspase-3 were effectively blocked by Bcl-2 overexpression. Caspase-9 knockdown failed to protect NCI-H460 cells from TRAIL-induced cell death, suggesting a minor role of this initiator caspase in this apoptotic pathway. Rather, knockdown of the XIAP antagonist Smac resulted in enhanced caspase-3 degradation after stimulation of cells with TRAIL. Of note, downregulation of XIAP had only limited effects on TRAIL sensitivity of wild-type NCI-H460 cells, but resensitized Bcl-2 overexpressing cells for TRAIL-induced apoptosis. In particular, XIAP knockdown in combination with TRAIL allowed the final cleavage step of caspase-3 to generate the catalytically active p17 fragment, whose production was otherwise blocked in Bcl-2 overexpressing cells. Together, our data strongly suggest that XIAP-mediated inhibition of final caspase-3 processing is the last and major hurdle in TRAIL-induced apoptosis in NCI-H460 cells, which can be overcome by Smac in a Bcl-2 level dependent manner. Quantitative investigation of the XIAP/Smac interplay using a mathematical model approach corroborates our experimental data strengthening the suggested roles of XIAP and Smac as critical determinants for TRAIL sensitivity

An individual-based simulation framework for dynamic, heterogeneous cell populations during extrinsic stimulations

status: publishe

Moderate Bcl-2 overexpression effectively protects NCI-H460 cells from TRAIL-induced apoptosis.

<p>(A) Expression of FLAG-tagged Bcl-2 in NCI-H460 cells. Equal amounts of whole cell lysates from NCI-H460 wild type and NCI-H460/Bcl-2 were analyzed by immunoblotting. Note that the tagged Bcl-2 protein appears at a somewhat lower molecular weight range as compared to wild-type Bcl-2. Equal loading was confirmed by re-probing the membrane with α-tubulin specific antibody. (B) Detection of Bcl-2 by flow cytometry. Fixed cells were permeabilized and stained with anti-Bcl-2 or corresponding isotype control primary antibody and PE-labeled secondary antibody. Cells were analyzed by flow cytometry. (C) Cytotoxicity assays of NCI-H460 and NCI-H460/Bcl-2 cells using Db-scTRAIL. Cells were treated with increasing concentrations of Db-scTRAIL for 24 h, viable cells were stained with crystal violet and quantified at 550 nm. All values were normalized to values from unstimulated cells. Shown are mean values ± SD calculated from triplicates. Data shown is representative of three independent experiments. (D) Db-scTRAIL induces apoptosis in NCI-H460. Cells were co-treated with Db-scTRAIL and z-VAD-fmk (20 μM) or only the carrier DMSO for 24 h. Viable, apoptotic (Annexin V-FITC-positive) and necrotic (PI-positive) cells were determined after flow cytometric analysis. (E) Db-scTRAIL induces cytochrome c and Smac release into the cytosol in wild-type NCI-H460 cells only. Cells were left untreated or treated with Db-scTRAIL (0.5 nM) for the indicated time periods. Cytosolic extracts were prepared and subjected to western blotting with cytochrome c and Smac specific antibodies. Equal loading was confirmed by reprobing the membrane with α-tubulin antibody. (F) ABT-737 resensitizes NCI-H460/Bcl-2 cells. Cells were preincubated with 2.5 μM ABT-737 alone (filled triangle) or in combination with 50 μM z-VAD-fmk (filled circles) or DMSO as control (open squares) and treated with Db-scTRAIL. Viable cells were quantified by crystal violet staining after 24 h. Data represents mean values ± SD calculated from triplicates and shown is a representative of three independent experiments.</p

XIAP downregulation restores TRAIL sensitivity in Bcl-2 overexpressing cells.

<p>(A) Downregulation of XIAP by siRNA treatment. Cells were left untreated (UT) or were transiently transfected with non-targeting (siControl) or siRNA specific for XIAP (siXIAP). Protein expression was determined 48 h post transfection from whole cell lysates <i>via</i> immunoblot assay using an XIAP specific antibody. As control c-IAP1 and survivin were included. (B) and (C) NCI-H460 wild type (B) and NCI-H460/Bcl-2 cells (C) had been pretreated as in (A) were treated with serial dilutions of Db-scTRAIL (open symbols). NCI-H460/Bcl-2 cells transfected with siXIAP were incubated with 50 μM z-VAD-fmk and 1 nM Db-scTRAIL (filled triangle). After 24 h viable cells were stained with crystal violet. All values were normalized to those from unstimulated cells. The experiment was performed in triplicates and the data shown are representative of three independent experiments. (D) NCI-H460/Bcl-2 cells had been pretreated as in (A) were treated with Db-scTRAIL (1 nM) for 2 and 4 h and analyzed by immunoblotting using antibodies for cleaved caspase-8, -9 and -3. Tubulin-α was used as loading control. Blot shown is representative of three independent experiments.</p