Identification of a set of genes potentially responsible for resistance to ferroptosis in lung adenocarcinoma cancer stem cells.

Scientific literature supports the evidence that cancer stem cells (CSCs) retain inside low reactive oxygen species (ROS) levels and are, therefore, less susceptible to cell death, including ferroptosis, a type of cell death dependent on iron-driven lipid peroxidation. A collection of lung adenocarcinoma (LUAD) primary cell lines derived from malignant pleural effusions (MPEs) of patients was used to obtain 3D spheroids enriched for stem-like properties. We observed that the ferroptosis inducer RSL3 triggered lipid peroxidation and cell death in LUAD cells when grown in 2D conditions; however, when grown in 3D conditions, all cell lines underwent a phenotypic switch, exhibiting substantial resistance to RSL3 and, therefore, protection against ferroptotic cell death. Interestingly, this phenomenon was reversed by disrupting 3D cells and growing them back in adherence, supporting the idea of CSCs plasticity, which holds that cancer cells have the dynamic ability to transition between a CSC state and a non-CSC state. Molecular analyses showed that ferroptosis resistance in 3D spheroids correlated with an increased expression of antioxidant genes and high levels of proteins involved in iron storage and export, indicating protection against oxidative stress and low availability of iron for the initiation of ferroptosis. Moreover, transcriptomic analyses highlighted a novel subset of genes commonly modulated in 3D spheroids and potentially capable of driving ferroptosis protection in LUAD-CSCs, thus allowing to better understand the mechanisms of CSC-mediated drug resistance in tumors.

ALDOC- and ENO2- driven glucose metabolism sustains 3D tumor spheroids growth regardless of nutrient environmental conditions: a multi-omics analysis.

BACKGROUND: Metastases are the major cause of cancer-related morbidity and mortality. By the time cancer cells detach from their primary site to eventually spread to distant sites, they need to acquire the ability to survive in non-adherent conditions and to proliferate within a new microenvironment in spite of stressing conditions that may severely constrain the metastatic process. In this study, we gained insight into the molecular mechanisms allowing cancer cells to survive and proliferate in an anchorage-independent manner, regardless of both tumor-intrinsic variables and nutrient culture conditions. METHODS: 3D spheroids derived from lung adenocarcinoma (LUAD) and breast cancer cells were cultured in either nutrient-rich or -restricted culture conditions. A multi-omics approach, including transcriptomics, proteomics, and metabolomics, was used to explore the molecular changes underlying the transition from 2 to 3D cultures. Small interfering RNA-mediated loss of function assays were used to validate the role of the identified differentially expressed genes and proteins in H460 and HCC827 LUAD as well as in MCF7 and T47D breast cancer cell lines. RESULTS: We found that the transition from 2 to 3D cultures of H460 and MCF7 cells is associated with significant changes in the expression of genes and proteins involved in metabolic reprogramming. In particular, we observed that 3D tumor spheroid growth implies the overexpression of ALDOC and ENO2 glycolytic enzymes concomitant with the enhanced consumption of glucose and fructose and the enhanced production of lactate. Transfection with siRNA against both ALDOC and ENO2 determined a significant reduction in lactate production, viability and size of 3D tumor spheroids produced by H460, HCC827, MCF7, and T47D cell lines. CONCLUSIONS: Our results show that anchorage-independent survival and growth of cancer cells are supported by changes in genes and proteins that drive glucose metabolism towards an enhanced lactate production. Notably, this finding is valid for all lung and breast cancer cell lines we have analyzed in different nutrient environmental conditions. broader Validation of this mechanism in other cancer cells of different origin will be necessary to broaden the role of ALDOC and ENO2 to other tumor types. Future in vivo studies will be necessary to assess the role of ALDOC and ENO2 in cancer metastasis.

B4GALT1 as a New Biomarker of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by progressive scarring of the lung that involves the pulmonary interstitium. The disease may rapidly progress, leading to respiratory failure, and the long-term survival is poor. There are no accurate biomarkers available so far. Our aim was to evaluate the expression of the B4GALT1 in patients with IPF. Analysis of B4GALT1 gene expression was performed in silico on two gene sets, retrieved from the Gene Expression Omnibus database. Expression of B4GALT1 was then evaluated, both at the mRNA and protein levels, on lung specimens obtained from lung biopsies of 4 IPF patients, on one IPF-derived human primary cell and on 11 cases of IPF associated with cancer. In silico re-analysis demonstrated that the B4GALT1 gene was overexpressed in patients and human cell cultures with IPF (p = 0.03). Network analysis demonstrated that B4GALT1 upregulation was correlated with genes belonging to the EMT pathway (p = 0.01). The overexpression of B4GALT1 was observed, both at mRNA and protein levels, in lung biopsies of our four IPF patients and in the IPF-derived human primary cell, in other fibrotic non-lung tissues, and in IPF associated with cancer. In conclusion, our results indicate that B4GALT1 is overexpressed in IPF and could represent a novel marker of this disease.

Deconvolution of malignant pleural effusions immune landscape unravels a novel macrophage signature associated with worse clinical outcome in lung adenocarcinoma patients.

BACKGROUND: Immune checkpoint inhibitors are still unable to provide clinical benefit to the large majority of non-small cell lung cancer (NSCLC) patients. A deeper characterization of the tumor immune microenvironment (TIME) is expected to shed light on the mechanisms of cancer immune evasion and resistance to immunotherapy. Here, we exploited malignant pleural effusions (MPEs) from lung adenocarcinoma (LUAD) patients as a model system to decipher TIME in metastatic NSCLC. METHODS: Mononuclear cells from MPEs (PEMC) and peripheral blood (PBMC), cell free pleural fluid and/or plasma were collected from a total of 24 LUAD patients and 12 healthy donors. Bulk-RNA sequencing was performed on total RNA extracted from PEMC and matched PBMC. The DEseq2 Bioconductor package was used to perform differential expression analysis and CIBERSORTx for the regression-based immune deconvolution of bulk gene expression data. Cytokinome analysis of cell-free pleural fluid and plasma samples was performed using a 48-Plex Assay panel. THP-1 monocytic cells were used to assess macrophage polarization. Survival analyses on NSCLC patients were performed using KM Plotter (LUAD, N=672; lung squamous cell carcinoma, N=271). RESULTS: Transcriptomic analysis of immune cells and cytokinome analysis of soluble factors in the pleural fluid depicted MPEs as a metastatic niche in which all the components required for an effective antitumor response are present, but conscripted in a wound-healing, proinflammatory and tumor-supportive mode. The bioinformatic deconvolution analysis revealed an immune landscape dominated by myeloid subsets with the prevalence of monocytes, protumoral macrophages and activated mast cells. Focusing on macrophages we identified an MPEs-distinctive signature associated with worse clinical outcome in LUAD patients. CONCLUSIONS: Our study reports for the first time a wide characterization of MPEs LUAD microenvironment, highlighting the importance of specific components of the myeloid compartment and opens new perspectives for the rational design of new therapies for metastatic NSCLC.

Sustained Systemic Levels of IL-6 Impinge Early Muscle Growth and Induce Muscle Atrophy and Wasting in Adulthood.

IL-6 is a pleiotropic cytokine that can exert different and opposite effects. The muscle-induced and transient expression of IL-6 can act in an autocrine or paracrine manner, stimulating anabolic pathways associated with muscle growth, myogenesis, and with regulation of energy metabolism. In contrast, under pathologic conditions, including muscular dystrophy, cancer associated cachexia, aging, chronic inflammatory diseases, and other pathologies, the plasma levels of IL-6 significantly increase, promoting muscle wasting. Nevertheless, the specific physio-pathological role exerted by IL-6 in the maintenance of differentiated phenotype remains to be addressed. The purpose of this study was to define the role of increased plasma levels of IL-6 on muscle homeostasis and the mechanisms contributing to muscle loss. Here, we reported that increased plasma levels of IL-6 promote alteration in muscle growth at early stage of postnatal life and induce muscle wasting by triggering a shift of the slow-twitch fibers toward a more sensitive fast fiber phenotype. These findings unveil a role for IL-6 as a potential biomarker of stunted growth and skeletal muscle wasting.

SCD1, autophagy and cancer: implications for therapy.

BACKGROUND: Autophagy is an intracellular degradation system that removes unnecessary or dysfunctional components and recycles them for other cellular functions. Over the years, a mutual regulation between lipid metabolism and autophagy has been uncovered. METHODS: This is a narrative review discussing the connection between SCD1 and the autophagic process, along with the modality through which this crosstalk can be exploited for therapeutic purposes. RESULTS: Fatty acids, depending on the species, can have either activating or inhibitory roles on autophagy. In turn, autophagy regulates the mobilization of fat from cellular deposits, such as lipid droplets, and removes unnecessary lipids to prevent cellular lipotoxicity. This review describes the regulation of autophagy by lipid metabolism in cancer cells, focusing on the role of stearoyl-CoA desaturase 1 (SCD1), the key enzyme involved in the synthesis of monounsaturated fatty acids. SCD1 plays an important role in cancer, promoting cell proliferation and metastasis. The role of autophagy in cancer is more complex since it can act either by protecting against the onset of cancer or by promoting tumor growth. Mounting evidence indicates that autophagy and lipid metabolism are tightly interconnected. CONCLUSION: Here, we discuss controversial findings of SCD1 as an autophagy inducer or inhibitor in cancer, highlighting how these activities may result in cancer promotion or inhibition depending upon the degree of cancer heterogeneity and plasticity.

IL-10, IL-13, Eotaxin and IL-10/IL-6 ratio distinguish breast implant-associated anaplastic large-cell lymphoma from all types of benign late seromas.

Breast implant-associated anaplastic large-cell lymphoma (BI-ALCL) is an uncommon peripheral T cell lymphoma usually presenting as a delayed peri-implant effusion. Chronic inflammation elicited by the implant has been implicated in its pathogenesis. Infection or implant rupture may also be responsible for late seromas. Cytomorphological examination coupled with CD30 immunostaining and eventual T-cell clonality assessment are essential for BI-ALCL diagnosis. However, some benign effusions may also contain an oligo/monoclonal expansion of CD30 + cells that can make the diagnosis challenging. Since cytokines are key mediators of inflammation, we applied a multiplexed immuno-based assay to BI-ALCL seromas and to different types of reactive seromas to look for a potential diagnostic BI-ALCL-associated cytokine profile. We found that BI-ALCL is characterized by a Th2-type cytokine milieu associated with significant high levels of IL-10, IL-13 and Eotaxin which discriminate BI-ALCL from all types of reactive seroma. Moreover, we found a cutoff of IL10/IL-6 ratio of 0.104 is associated with specificity of 100% and sensitivity of 83% in recognizing BI-ALCL effusions. This study identifies promising biomarkers for initial screening of late seromas that can facilitate early diagnosis of BI-ALCL.

B4GALT1 Is a New Candidate to Maintain the Stemness of Lung Cancer Stem Cells.

BACKGROUND: According to the cancer stem cells (CSCs) hypothesis, a population of cancer cells with stem cell properties is responsible for tumor propagation, drug resistance, and disease recurrence. Study of the mechanisms responsible for lung CSCs propagation is expected to provide better understanding of cancer biology and new opportunities for therapy. METHODS: The Lung Adenocarcinoma (LUAD) NCI-H460 cell line was grown either as 2D or as 3D cultures. Transcriptomic and genome-wide chromatin accessibility studies of 2D vs. 3D cultures were carried out using RNA-sequencing and Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq), respectively. Reverse transcription polymerase chain reaction (RT-PCR) was also carried out on RNA extracted from primary cultures derived from malignant pleural effusions to validate RNA-seq results. RESULTS: RNA-seq and ATAC-seq data disentangled transcriptional and genome accessibility variability of 3D vs. 2D cultures in NCI-H460 cells. The examination of genomic landscape of genes upregulated in 3D vs. 2D cultures led to the identification of 2D cultures led to the identification of Beta-1,4-galactosyltranferase 1 (B4GALT1) as the top candidate. B4GALT1 as the top candidate. B4GALT1 was validated as a stemness factor, since its silencing caused strong inhibition of 3D spheroid formation. CONCLUSION: Combined transcriptomic and chromatin accessibility study of 3D vs. 2D LUAD cultures led to the identification of B4GALT1 as a new factor involved in the propagation and maintenance of LUAD CSCs.

Effects of IGF-1 isoforms on muscle growth and sarcopenia.

The decline in skeletal muscle mass and strength occurring in aging, referred as sarcopenia, is the result of many factors including an imbalance between protein synthesis and degradation, changes in metabolic/hormonal status, and in circulating levels of inflammatory mediators. Thus, factors that increase muscle mass and promote anabolic pathways might be of therapeutic benefit to counteract sarcopenia. Among these, the insulin-like growth factor-1 (IGF-1) has been implicated in many anabolic pathways in skeletal muscle. IGF-1 exists in different isoforms that might exert different role in skeletal muscle. Here we study the effects of two full propeptides IGF-1Ea and IGF-1Eb in skeletal muscle, with the aim to define whether and through which mechanisms their overexpression impacts muscle aging. We report that only IGF-1Ea expression promotes a pronounced hypertrophic phenotype in young mice, which is maintained in aged mice. Nevertheless, examination of aged transgenic mice revealed that the local expression of either IGF-1Ea or IGF-1Eb transgenes was protective against age-related loss of muscle mass and force. At molecular level, both isoforms activate the autophagy/lysosome system, normally altered during aging, and increase PGC1-α expression, modulating mitochondrial function, ROS detoxification, and the basal inflammatory state occurring at old age. Moreover, morphological integrity of neuromuscular junctions was maintained and preserved in both MLC/IGF-1Ea and MLC/IGF-1Eb mice during aging. These data suggest that IGF-1 is a promising therapeutic agent in staving off advancing muscle weakness.

Skeletal muscle myopenia in mice model of bile duct ligation and carbon tetrachloride-induced liver cirrhosis.

Skeletal muscle myopathy is universal in cirrhotic patients, however, little is known about the main mechanisms involved. The study aims to investigate skeletal muscle morphological, histological, and functional modifications in experimental models of cirrhosis and the principal molecular pathways responsible for skeletal muscle myopathy. Cirrhosis was induced by bile duct ligation (BDL) and carbon tetrachloride (CCl4) administration in mice. Control animals (CTR) underwent bile duct exposure or vehicle administration only. At sacrifice, peripheral muscles were dissected and weighed. Contractile properties of extensor digitorum longus (EDL) were studied in vitro. Muscle samples were used for histological and molecular analysis. Quadriceps muscle histology revealed a significant reduction in cross-sectional area of muscle and muscle fibers in cirrhotic mice with respect to CTR. Kinetic properties of EDL in both BDL and CCl4 were reduced with respect to CTR; BDL mice also showed a reduction in muscle force and a decrease in the resistance to fatigue. Increase in myostatin expression associated with a decrease in AKT-mTOR expressions was observed in BDL mice, together with an increase in LC3 protein levels. Upregulation of the proinflammatory citochines TNF-a and IL6 and an increased expression of NF-kB and MuRF-1 were observed in CCl4 mice. In conclusion, skeletal muscle myopenia was present in experimental models of BDL and CCl4-induced cirrhosis. Moreover, reduction in protein synthesis and activation of protein degradation were the main mechanisms responsible for myopenia in BDL mice, while activation of ubiquitin-pathway through inflammatory cytokines seems to be the main potential mechanism involved in CCl4 mice.