Ferroptosis constitutes a promising therapeutic strategy against cancer by efficiently targeting the highly tumorigenic and treatment-resistant cancer stem cells (CSCs). We previously showed that the lysosomal iron-targeting drug Salinomycin (Sal) was able to eliminate CSCs by triggering ferroptosis. Here, in a well-established breast CSCs model (human mammary epithelial HMLER CD24^low/CD44^high), we identified that pharmacological inhibition of the mechanistic target of rapamycin (mTOR), suppresses Sal-induced ferroptosis. Mechanistically, mTOR inhibition modulates iron cellular flux and thereby limits iron-mediated oxidative stress. Furthermore, integration of multi-omics data identified mitochondria as a key target of Sal action, leading to profound functional and structural alteration prevented by mTOR inhibition. On top of that, we found that Sal-induced metabolic plasticity is mainly dependent on the mTOR pathway. Overall, our findings provide experimental evidence for the mechanisms of mTOR as a crucial effector of Sal-induced ferroptosis pointing not only that metabolic reprogramming regulates ferroptosis, but also providing proof-of-concept that careful evaluation of such combination therapy (here mTOR and ferroptosis co-targeting) is required in the development of an effective treatment.

Accumulation of senescent dermal fibroblasts drives skin aging. The reactivation of proliferation is one strategy to modulate cell senescence. Recently, we reported the exact chemical composition of the hydrophilic extract of Oenothera biennis cell cultures (ObHEx) and we showed its skin anti-aging properties. The aim of this work is to assess its biological effect specifically on cell senescence. ObHEx action has been evaluated on normal human dermal fibroblasts subjected to stress-induced premature senescence (SIPS) through an ultra-deep proteomic analysis, leading to the most global senescence-associated proteome so far. Mass spectrometry data show that the treatment with ObHEx re-establishes levels of crucial mitotic proteins, strongly downregulated in senescent cells. To validate our proteomics findings, we proved that ObHEx can, in part, restore the activity of ‘senescence-associated-ß-galactosidase’, the most common hallmark of senescent cells. Furthermore, to assess if the upregulation of mitotic protein levels translates into a cell cycle re-entry, FACS experiments have been carried out, demonstrating a small but significative reactivation of senescent cell proliferation by ObHEx. In conclusion, the deep senescence-associated global proteome profiling published here provides a panel of hundreds of proteins deregulated by SIPS that can be used by the community to further understand senescence and the effect of new potential modulators. Moreover, proteomics analysis pointed to a specific promitotic effect of ObHEx on senescent cells. Thus, we suggest ObHEx as a powerful adjuvant against senescence associated with skin aging.

Despite the well-described association of skin lesions with Staphylococcus aureus , the distinct ability of clinical isolates to influence the local and systemic inflammatory response in a patient-specific manner is insufficiently characterized. In this study, we analyzed clinical recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by wounds chronically colonized with S. aureus , to explore the relationship between inflammatory immune response and strain diversity. Children with RDEB (moderate phenotype, n = 5; severe phenotype, n = 10) and controls ( n = 18) were enrolled in the study. Profiling of plasma proteins ( n = 800), immune cells ( n = 30 subsets and cytokine-producing cells), and cytokines ( n = 38) identified a specific inflammatory signature in severe disease. Furthermore, patients with severe RDEB presented a high frequency of interleukin-17A+ (IL-17A+) cells among CD4+ and mucosal-associated invariant T (MAIT) lymphocytes. Positive S. aureus cultures from the skin of patients with RDEB allowed whole-genome sequencing of patient strains and assessment of primary keratinocyte immune response upon bacterial challenge. S. aureus secretome and conditioned medium from keratinocytes challenged with S. aureus strains from patients with severe but not from those with moderate RDEB promoted strong activation and a pro–IL-17 response in both CD4+ and MAIT cells. Our findings show that S. aureus strains isolated from patients with severe RDEB induce an IL-17–skewed immune response and pave the way for precision microbiology to explain and predict the highly variable virulence potential of bacterial clinical isolates.

Recent evidence implicates altered RNA editing and dysregulated type I IFN signaling in immune-mediated diseases, including psoriasis, although the underlying genetic mechanisms remain poorly defined. We investigated four unrelated multiplex families with early-onset plaque psoriasis, with or without psoriatic arthritis, segregating as a monogenic trait and characterized by a strong IFN signature in skin and blood. Whole-exome sequencing identified four rare heterozygous loss-of-function mutations in ADAR1 cosegregating with disease and elevated IFN-stimulated gene expression. Six additional rare variants were detected in an independent cohort of 125 psoriasis patients. Single-cell transcriptomics identified keratinocytes and melanocytes as major IFN sources. Functional studies showed that ADAR1 knockdown or expression of ADAR1G1119R and ADAR1P3A alleles pathogenic variants reduced adenosine-to-inosine RNA editing and increased IFN-stimulated genes and inflammatory cytokines, effects reversed by upadacitinib and deucravacitinib. These findings define a novel IFN-dependent psoriasis subtype caused by inborn defects of ADAR1-mediated RNA editing, with direct implications for precision medicine in psoriatic disease.

Insulin-degrading enzyme is a zinc metalloprotease that degrades low-molecular-weight substrates, including insulin. Ubiquitous expression, high evolutionary conservation, upregulation of Ide in stress situations, and literature findings suggest a broader function of Ide in cell physiology and protein homeostasis that remains to be elucidated. We used proteomics and transcriptomics approaches to search for leads related to a broader role of Ide in protein homeostasis. We combined an analysis of the proteome and single-cell transcriptome of Ide^+/+ and Ide^−/− pancreatic islet cells with an examination of the interactome of human cytosolic Ide using proximity biotinylation. We observe an upregulation of pathways related to RNA processing, translation and splicing in Ide^+/+ relative to Ide^−/− islet cells. Corroborating these results and providing a potential mechanistic explanation, proximity biotinylation reveals interaction of Ide with several subunits of CCR4-NOT, a key mRNA deadenylase regulating gene expression “from birth to death”. We propose a speculative model in which human and murine Ide cooperate with CCR4-NOT to control protein expression in proteotoxic and metabolic stress situations through cooperation between their deadenylase and protease functions.

Pachyonychia congenita (PC) is a rare and painful skin disorder caused by dominant pathogenic variants in keratin genes (KRT6A/KRT6B/KRT6C/KRT16/KRT17), with no effective treatment. We developed a scalable, in-depth and miniaturized MS-based proteomics and phospho-proteomics analysis of full-thickness skin biopsies applied to 10 PC patients to elucidate pathogenic mechanisms and pinpoint therapeutic targets. We quantified 7200 Protein Groups (PGs) on average from 2mm snap-frozen skin samples, the most in-depth proteome coverage reported to date from a single-shot MS analysis. Among the identified PGs, ∼1400 proteins were differentially abundant in lesional vs. non-lesional samples. Enrichment analysis points towards impaired mitochondrial function, hyper-keratinization, enhanced immune response and a significant increase in cholesterol biosynthesis. Phosphoproteomics revealed hyper-phosphorylation of specific sites in PC-related keratins, confirmed the hyper-activation of EGFR and downstream kinases, including PKC and Src, and disclosed p38 MAPK activation. All of these kinases have been reported to phosphorylate keratins. Our study expands upon current understanding of the consequences of EGFR pathway activation, including increased cholesterol biosynthesis, thereby renewing interest in the use of statins for PC. Above all, it provides a solid foundation for the continued exploration of EGFR inhibitors and offers therapeutic avenues, particularly those using multikinase inhibitors.