Resorcinol alleviates alpha-terpineol-induced cell death in Schizosaccharomyces pombe via increased activity of the antioxidant enzyme Sod2.

Alpha-terpineol, popular monoterpenoid alcohol, is known to cause cytotoxicity in a few cancer cells or to have antioxidant activity, but underlying mechanisms or apoptotic processes in yeast cell death should be understood. We used the fission yeast (Schizosaccharomyces pombe) as a unicellular model to monitor cellular toxicology and physiological mechanisms for the involvement of alpha-terpineol in cell death. Alpha-terpineol caused Reactive oxygen species (ROS) overproduction and following cytotoxicity and apoptosis in a dose-dependent manner. The effect of oxidative stress was proved using sod1 and sod2 mutants (antioxidant-limited cells), and the results showed that apoptosis was caused by alpha-terpineol-driven oxidation. In addition, resorcinol, a herbal extract from medicinal plants, showed protective activity against alpha-terpineol cytotoxicity. Survival rates, apoptotic cell death ratios, oxidation levels, and antioxidant gene expressions were completely altered; surprisingly sod1 and sod2 levels dramatically increased. However, sod2 was highly upregulated in response to resorcinol treatment with alpha-terpineol. The potential role of the Sod2 enzyme was proved using sod2 mutant cells that do not have a mitochondrial radical-clearing activity. Consequently, the dose-dependent and ROS-mediated cytotoxic/apoptotic effects of alpha-terpineol and the Sod2-dependent protective and antioxidant effects of resorcinol were demonstrated in unicellular model organism S. pombe by this study.

PRDX6 contributes to selenocysteine metabolism and ferroptosis resistance.

Selenocysteine (Sec) metabolism is crucial for cellular function and ferroptosis prevention and has traditionally been thought to begin with the uptake of the Sec carrier selenoprotein P (SELENOP). Following uptake, Sec released from SELENOP undergoes metabolisation via selenocysteine lyase (SCLY), producing selenide, a substrate used by selenophosphate synthetase 2 (SEPHS2), which provides the essential selenium donor - selenophosphate - for the biosynthesis of the selenocysteine tRNA. Here, we report the discovery of an alternative pathway mediating Sec metabolisation that is independent of SCLY and mediated by peroxiredoxin 6 (PRDX6). Mechanistically, we demonstrate that PRDX6 can readily react with selenide and interact with SEPHS2, potentially acting as a selenium delivery system. Moreover, we demonstrate the presence and functional significance of this alternative route in cancer cells where we reveal a notable association between elevated expression of PRDX6 with a highly aggressive neuroblastoma subtype. Altogether, our study sheds light on a previously unrecognized aspect of Sec metabolism and its implications in ferroptosis, offering new avenues for therapeutic exploitation.

Low level of antioxidant capacity biomarkers but not target overexpression predicts vulnerability to ROS-inducing drugs.

Despite a strong rationale for why cancer cells are susceptible to redox-targeting drugs, such drugs often face tumor resistance or dose-limiting toxicity in preclinical and clinical studies. An important reason is the lack of specific biomarkers to better select susceptible cancer entities and stratify patients. Using a large panel of lung cancer cell lines, we identified a set of "antioxidant-capacity" biomarkers (ACB), which were tightly repressed, partly by STAT3 and STAT5A/B in sensitive cells, rendering them susceptible to multiple redox-targeting and ferroptosis-inducing drugs. Contrary to expectation, constitutively low ACB expression was not associated with an increased steady state level of reactive oxygen species (ROS) but a high level of nitric oxide, which is required to sustain high replication rates. Using ACBs, we identified cancer entities with a high percentage of patients with favorable ACB expression pattern, making it likely that more responders to ROS-inducing drugs could be stratified for clinical trials.

LRP8-mediated selenocysteine uptake is a targetable vulnerability in MYCN-amplified neuroblastoma.

Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR-activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN-amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc- . The identification of LRP8 as a specific vulnerability of MYCN-amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet-unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high-risk neuroblastoma and potentially other MYCN-amplified entities.

nNOS-derived NO modulates force production and iNO-derived NO the excitability in C2C12-derived 3D tissue engineering skeletal muscle via different NO signaling pathways.

Nitric oxide (NO) is a bioactive gas produced by one of the three NO synthases: neuronal NOS (nNOS), inducible (iNOS), and endothelial NOS (eNOS). NO has a relevant modulatory role in muscle contraction; this takes place through two major signaling pathways: (i) activation of soluble guanylate cyclase and, thus, protein kinase G or (ii) nitrosylation of sulfur groups of cysteine. Although it has been suggested that nNOS-derived NO is the responsible isoform in muscle contraction, the roles of eNOS and iNOS and their signaling pathways have not yet been clarified. To elucidate the action of each pathway, we optimized the generation of myooids, an engineered skeletal muscle tissue based on the C2C12 cell line. In comparison with diaphragm strips from wild-type mice, 180 myooids were analyzed, which expressed all relevant excitation-contraction coupling proteins and both nNOS and iNOS isoforms. Along with the biochemical results, myooids treated with NO donor (SNAP) and unspecific NOS blocker (L-NAME) revealed a comparable NO modulatory effect on force production as was observed in the diaphragm strips. Under the effects of pharmacological tools, we analyzed the myooids in response to electrical stimulation of two possible signaling pathways and NO sources. The nNOS-derived NO exerted its negative effect on force production via the sGG-PKG pathway, while iNOS-derived NO increased the excitability in response to sub-threshold electrical stimulation. These results strengthen the hypotheses of previous reports on the mechanism of action of NO during force production, showed a novel function of iNOS-derived NO, and establish the myooid as a novel and robust alternative model for pathophysiological skeletal muscle research.

High-throughput functional characterization of protein phosphorylation sites in yeast.

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, fewer than 5% of thousands of recently discovered phosphosites have been functionally annotated. In this study, we devised a chemical genetic approach to study the functional relevance of phosphosites in Saccharomyces cerevisiae. We generated 474 yeast strains with mutations in specific phosphosites that were screened for fitness in 102 conditions, along with a gene deletion library. Of these phosphosites, 42% exhibited growth phenotypes, suggesting that these are more likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions and validated a subset by thermal proteome profiling and lipidomics. A high fraction exhibited phenotypes not seen in the corresponding gene deletion, suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This high-throughput approach allows for functionally characterizing individual phosphosites at scale.

MYCN mediates cysteine addiction and sensitizes neuroblastoma to ferroptosis.

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors.

Systematic dissection of transcriptional regulatory networks by genome-scale and single-cell CRISPR screens.

Millions of putative transcriptional regulatory elements (TREs) have been cataloged in the human genome, yet their functional relevance in specific pathophysiological settings remains to be determined. This is critical to understand how oncogenic transcription factors (TFs) engage specific TREs to impose transcriptional programs underlying malignant phenotypes. Here, we combine cutting edge CRISPR screens and epigenomic profiling to functionally survey ≈15,000 TREs engaged by estrogen receptor (ER). We show that ER exerts its oncogenic role in breast cancer by engaging TREs enriched in GATA3, TFAP2C, and H3K27Ac signal. These TREs control critical downstream TFs, among which TFAP2C plays an essential role in ER-driven cell proliferation. Together, our work reveals novel insights into a critical oncogenic transcription program and provides a framework to map regulatory networks, enabling to dissect the function of the noncoding genome of cancer cells.

PAX8 activates metabolic genes via enhancer elements in Renal Cell Carcinoma.

Transcription factor networks shape the gene expression programs responsible for normal cell identity and pathogenic state. Using Core Regulatory Circuitry analysis (CRC), we identify PAX8 as a candidate oncogene in Renal Cell Carcinoma (RCC) cells. Validation of large-scale functional genomic screens confirms that PAX8 silencing leads to decreased proliferation of RCC cell lines. Epigenomic analyses of PAX8-dependent cistrome demonstrate that PAX8 largely occupies active enhancer elements controlling genes involved in various metabolic pathways. We selected the ferroxidase Ceruloplasmin (CP) as an exemplary gene to dissect PAX8 molecular functions. PAX8 recruits histone acetylation activity at bound enhancers looping onto the CP promoter. Importantly, CP expression correlates with sensitivity to PAX8 silencing and identifies a subset of RCC cases with poor survival. Our data identifies PAX8 as a candidate oncogene in RCC and provides a potential biomarker to monitor its activity.

Detection of the water reservoir in a forming planetary system.

Icy bodies may have delivered the oceans to the early Earth, yet little is known about water in the ice-dominated regions of extrasolar planet-forming disks. The Heterodyne Instrument for the Far-Infrared on board the Herschel Space Observatory has detected emission lines from both spin isomers of cold water vapor from the disk around the young star TW Hydrae. This water vapor likely originates from ice-coated solids near the disk surface, hinting at a water ice reservoir equivalent to several thousand Earth oceans in mass. The water's ortho-to-para ratio falls well below that of solar system comets, suggesting that comets contain heterogeneous ice mixtures collected across the entire solar nebula during the early stages of planetary birth.