Induction of stearoyl-CoA desaturase confers cell density-dependent ferroptosis resistance in melanoma.

Ferroptosis is a form of regulated cell death that is induced by inhibiting glutathione peroxidase 4 (GPX4), which eliminates lipid peroxidation. Ferroptosis induction is influenced by the cell environment. However, the cellular states altering ferroptosis susceptibility remain largely unknown. We found that melanoma cell lines became resistant to ferroptosis as cell density increased. Comparative transcriptome and metabolome analyses revealed that cell density-dependent ferroptosis resistance was coupled with a shift toward a lipogenic phenotype accompanied by strong induction of stearoyl-CoA desaturase (SCD). Database analysis of gene dependency across hundreds of cancer cell lines uncovered a negative correlation between GPX4 and SCD dependency. Importantly, SCD inhibition, either pharmacologically or through genetic knockout, sensitized melanoma cells to GPX4 inhibition, thereby attenuating ferroptosis resistance in cells at high density. Our findings indicate that transition to an SCD-inducing, lipogenic cell state produces density-dependent resistance to ferroptosis, which may provide a therapeutic strategy against melanoma.

Spautin-1 inhibits mitochondrial complex I and leads to suppression of the unfolded protein response and cell survival during glucose starvation.

The unfolded protein response (UPR) is an adaptive stress response pathway that is essential for cancer cell survival under endoplasmic reticulum stress such as during glucose starvation. In this study, we identified spautin-1, an autophagy inhibitor that suppresses ubiquitin-specific peptidase 10 (USP10) and USP13, as a novel UPR inhibitor under glucose starvation conditions. Spautin-1 prevented the induction of UPR-associated proteins, including glucose-regulated protein 78, activating transcription factor 4, and a splicing variant of x-box-binding protein-1, and showed preferential cytotoxicity in glucose-starved cancer cells. However, USP10 and USP13 silencing and treatment with other autophagy inhibitors failed to result in UPR inhibition and preferential cytotoxicity during glucose starvation. Using transcriptome and chemosensitivity-based COMPARE analyses, we identified a similarity between spautin-1 and mitochondrial complex I inhibitors and found that spautin-1 suppressed the activity of complex I extracted from isolated mitochondria. Our results indicated that spautin-1 may represent an attractive mitochondria-targeted seed compound that inhibits the UPR and cancer cell survival during glucose starvation.

Neuronal DAF-16-to-intestinal DAF-16 communication underlies organismal lifespan extension in C. elegans.

Previous studies have revealed the importance of inter-tissue communications for lifespan regulation. However, the inter-tissue network responsible for lifespan regulation is not well understood, even in a simple organism Caenorhabditis elegans. To understand the mechanisms underlying systemic lifespan regulation, we focused on lifespan regulation by the insulin/insulin-like growth factor-1 signaling (IIS) pathway; IIS reduction activates the DAF-16/FOXO transcription factor, which results in lifespan extension. Our tissue-specific knockdown and knockout analyses demonstrated that IIS reduction in neurons and the intestine markedly extended lifespan. DAF-16 activation in neurons resulted in DAF-16 activation in the intestine and vice versa. Our dual gene manipulation method revealed that intestinal and neuronal DAF-16 mediate longevity induced by daf-2 knockout in neurons and the intestine, respectively. In addition, the systemic regulation of intestinal DAF-16 required the IIS pathway in intestinal and neurons. Collectively, these results highlight the importance of the neuronal DAF-16-to-intestinal DAF-16 communication for organismal lifespan regulation.

Disrupting ATF4 Expression Mechanisms Provides an Effective Strategy for BRAF-Targeted Melanoma Therapy.

BRAF V600 mutation influences cellular signaling pathways for melanoma development. However, the role of oncogenic BRAF in adaptive stress response pathways is not fully understood. Here, we show that oncogenic BRAF plays an essential role in the induction of ATF4 following the activation of general control non-derepressible 2 (GCN2) kinase during nutrient stress and BRAF-targeted, therapeutic stress. Under GCN2 activation, BRAF ensures ATF4 induction by utilizing mTOR and eIF4B as downstream regulators. In contrast to the MEK-ERK pathway, this signaling pathway remains temporarily active even during treatment with BRAF inhibitors, thereby enabling the transient induction of ATF4. We also identify a chemical compound that prevents BRAF inhibitor-induced activation of the GCN2-ATF4 pathway and produces synergistic cell killing with BRAF inhibitors. Our findings establish a collaborative relationship between oncogenic BRAF and the GCN2-ATF4 signaling pathway, which may provide a novel therapeutic approach to target the adaptive stress response.

The kinase PERK represses translation of the G-protein-coupled receptor LGR5 and receptor tyrosine kinase ERBB3 during ER stress in cancer cells.

As a branch of the unfolded protein response, protein kinase R-like endoplasmic reticulum kinase (PERK) represses global translation in response to endoplasmic reticulum (ER) stress. This pathophysiological condition is associated with the tumor microenvironment in cancer. Previous findings in our lab have suggested that PERK selectively represses translation of some mRNAs, but this possibility awaits additional investigation. In this study, we show that a stem-cell marker protein, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), is rapidly depleted in colon cancer cells during ER stress, an effect that depended on the PERK-mediated translational repression. Indeed, the PERK inhibition led to the accumulation of premature, underglycosylated forms of LGR5, which were produced only at low levels during proper PERK activation. Unlike the mature LGR5 form, which is constitutively degraded regardless of PERK activation, the underglycosylated LGR5 exhibited a prolonged half-life and accumulated inside the cells without being expressed on the cell surface. We also found that Erb-B2 receptor tyrosine kinase 3 (ERBB3) is subjected to a similarly-regulated depletion by PERK, whereas the epidermal growth factor receptor (EGFR), stress-inducible heat-shock protein family A (Hsp70) member 5 (HSPA5), and anterior gradient 2 protein-disulfide isomerase family member (AGR2) were relatively. insensitive to the PERK-mediated repression of translation. These results indicate that LGR5 and ERBB3 are targets for PERK-mediated translational repression during ER stress.

InDePTH: detection of hub genes for developing gene expression networks under anticancer drug treatment.

It has been difficult to elucidate the structure of gene regulatory networks under anticancer drug treatment. Here, we developed an algorithm to highlight the hub genes that play a major role in creating the upstream and downstream relationships within a given set of differentially expressed genes. The directionality of the relationships between genes was defined using information from comprehensive collections of transcriptome profiles after gene knockdown and overexpression. As expected, among the drug-perturbed genes, our algorithm tended to derive plausible hub genes, such as transcription factors. Our validation experiments successfully showed the anticipated activity of certain hub gene in establishing the gene regulatory network that was associated with cell growth inhibition. Notably, giving such top priority to the hub gene was not achieved by ranking fold change in expression and by the conventional gene set enrichment analysis of drug-induced transcriptome data. Thus, our data-driven approach can facilitate to understand drug-induced gene regulatory networks for finding potential functional genes.

Mitochondrial deficiency impairs hypoxic induction of HIF-1 transcriptional activity and retards tumor growth.

Mitochondria can be involved in regulating cellular stress response to hypoxia and tumor growth, but little is known about that mechanistic relationship. Here, we show that mitochondrial deficiency severely retards tumor xenograft growth with impairing hypoxic induction of HIF-1 transcriptional activity. Using mtDNA-deficient ρ0 cells, we found that HIF-1 pathway activation was comparable in slow-growing ρ0 xenografts and rapid-growing parental xenografts. Interestingly, we found that ex vivo ρ0 cells derived from ρ0 xenografts exhibited slightly increased HIF-1α expression and modest HIF-1 pathway activation regardless of oxygen concentration. Surprisingly, ρ0 cells, as well as parental cells treated with oxidative phosphorylation inhibitors, were unable to boost HIF-1 transcriptional activity during hypoxia, although HIF-1α protein levels were ordinarily increased in these cells under hypoxic conditions. These findings indicate that mitochondrial deficiency causes loss of hypoxia-induced HIF-1 transcriptional activity and thereby might lead to a constitutive HIF-1 pathway activation as a cellular adaptation mechanism in tumor microenvironment.

PMEPA1, a TGF-ß- and hypoxia-inducible gene that participates in hypoxic gene expression networks in solid tumors.

Prostate transmembrane protein, androgen induced 1 (PMEPA1) is highly expressed in various solid tumors and is known to play important roles in the transforming growth factor-ß (TGF-ß) signaling pathway. Here, we demonstrate a novel relationship between PMEPA1 and hypoxia, a common microenvironmental stress condition in solid tumors. We showed that induction of PMEPA1 expression occurred during hypoxia in a manner dependent on both TGF-ß signaling and hypoxia-inducible factor-1 (HIF-1) pathways. Furthermore, overexpression and knockdown experiments revealed that PMEPA1 enhanced HIF-1 transcription activity. Bioinformatics analyses of PMEPA1-correlated genes using a gene expression database in clinical settings showed significant enrichment of gene sets defined by TGF-ß and hypoxia and these two signaling pathways-related angiogenesis and epithelial-mesenchymal transition in many types of solid tumors. Collectively, our findings indicated that PMEPA1 participates in TGF-ß- and hypoxia-regulated gene expression networks in solid tumors and thereby may contribute to tumor progression.

Light transmission of the ocular media in birds and mammals.

Differences in the ultraviolet (UV) cutoff of ocular media between birds and mammals have been revealed by spectrophotometric measurements of the transmission of light wavelengths by the cornea, lens and vitreous body in chickens, crows, quails, rats, rabbits and pigs. The light transmission values of the cornea were shown to be above 50% for wavelengths of 330-800 nm in birds, 300-800 nm in rat and 310-800 nm in mammals except for rat. For the lens, the light transmission values were shown to be above 50% for wavelengths of 320-800 nm in birds and rat and 390-800 nm in mammals except for rat. Thus, among the ocular media, the cornea in birds and the lens in mammals except for rat may play a role as a major UV cutoff filter.

Population abundance of potentially pathogenic organisms in intestinal microbiome of jungle crow (Corvus macrorhynchos) shown with 16S rRNA gene-based microbial community analysis.

Jungle Crows (Corvus macrorhynchos) prefer human habitats because of their versatility in feeding accompanied with human food consumption. Therefore, it is important from a public health viewpoint to characterize their intestinal microbiota. However, no studies have been involved in molecular characterization of the microbiota based on huge and reliable number of data acquisition. In this study, 16S rRNA gene-based microbial community analysis coupled with the next-generation DNA sequencing techniques was applied to the taxonomic classification of intestinal microbiome for three jungle crows. Clustering of the reads into 130 operational taxonomic units showed that at least 70% of analyzed sequences for each crow were highly homologous to Eimeria sp., which belongs to the protozoan phylum Apicomplexa. The microbiotas of three crows also contained potentially pathogenic bacteria with significant percentages, such as the genera Campylobacter and Brachyspira. Thus, the profiling of a large number of 16S rRNA gene sequences in crow intestinal microbiomes revealed the high-frequency existence or vestige of potentially pathogenic microorganisms.

High levels of apolipoproteins found in the soluble fraction of avian cornea.

Water-soluble proteins in avian corneas were profiled by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Comparative protein profiling of avian and mammalian corneas revealed five major protein spots specifically detected in avian species. These proteins were identified as apolipoproteins A1 and D by tandem mass spectrometry sequencing. This is the first report of the presence of apolipoproteins in avian cornea. These results could provide insight into the role of lipid metabolism in the avian-specific function of cornea.

Microstructure characteristics of the cornea in birds and mammals.

In this study, the microstructure of the cornea was compared among chickens (Gallus gallus), jungle crows (Corvus macrorhynchos), rats (Rattus norvegicus) and rabbits (Oryctolagus cuniculus). The density of keratocytes in the mammals was over 3 times that in the birds. The size of the keratocytes in the birds and rat were significantly lower than those in the rabbit. Using scanning and transmission electron microscopy, the bundles of collagen fibers in the birds were found to be well arranged, while those in the mammals were arranged randomly. The collagen lamellae of the birds were significantly thicker than those of the mammals, and the numbers of collagen lamellae in the birds were significantly smaller than in the mammals. The center-to-center distances between the collagen fibrils of the chicken and rabbit were significantly larger than those of the crow and rat. The densities of collagen fibrils in the chicken and rabbit were significantly less than those of the crow and rat.

Sensitive fluorescent microplate bioassay using recombinant Escherichia coli with multiple promoter-reporter units in tandem for detection of arsenic.

Genetically modified bacterial biosensors can detect specific environmental compounds. Here, we attempted to establish a fluorescent microplate method to detect arsenic using recombinant Escherichia coli cells transformed with plasmids harboring three tandem copies of the ars promoter/operator-the gene for green fluorescent protein (gfp). In the biosensors, one copy of arsR, whose transcription is autoregulated by the ars promoter/operator and ArsR in the genome of E. coli, was placed in trans in another plasmid under the control of isopropyl-1-thio-beta-D-galactopyranoside-inducible promoter. First, this manipulation enabled regulation of the arsR expression at an adequate level. Second, the copy number of reporter unit also affected signal and noise. When the plasmid harboring three copies of the reporter unit was used, the signal-to-noise ratio doubled and the detection limit decreased from 20 to 7.5 microg L(-1) As(III), compared to the use of the plasmid harboring one copy of the ars promoter/operator-arsR-gfp. Thus, segregation of arsR from the ars promoter/operator-gfp using two plasmids is effective in regulating the signal-to-noise ratio and the detection limit with the different functions.