Increased mTOR Signaling and Impaired Autophagic Flux Are Hallmarks of SARS-CoV-2 Infection.

The COVID-19 (Coronavirus Disease 2019), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), severely affects mainly individuals with pre-existing comorbidities. Here our aim was to correlate the mTOR (mammalian/mechanistic Target of Rapamycin) and autophagy pathways with the disease severity. Through western blotting and RNA analysis, we found increased mTOR signaling and suppression of genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2 as well as in transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients. Immunofluorescence co-localization assays also indicated that SARS-CoV-2 colocalizes within autophagosomes but not with a lysosomal marker. Our findings indicate that SARS-CoV-2 can benefit from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling.

Resistance exercise attenuates IKKε phosphorylation and hepatic fat accumulation of obese mice.

Obesity is associated with low-grade inflammation and disturbances in hepatic metabolism. This study aimed to investigate the effects of resistance exercise on inflammatory signalling related to IκB kinase (IKK) ɛ protein (IKKɛ) and on hepatic fat accumulation in obese mice. Male Swiss mice were distributed into three groups: control (CTL) fed with standard chow; obese (OB) mice induced by a high-fat diet (HFD); obese exercised (OB + RE) mice fed with HFD and submitted to a resistance exercise training. The resistance exercise training protocol consisted of 20 sets/3 ladder climbs for 8 weeks, three times/week on alternate days. The training overload was equivalent to 70% of the maximum load supported by the rodent. Assays were performed to evaluate weight gain, hepatic fat content, fasting glucose, insulin sensitivity, IKKɛ phosphorylation and proteins related to insulin signalling and lipogenesis in the liver. Mice that received the high-fat diet showed greater adiposity, impaired insulin sensitivity, increased fasting glucose and increased hepatic fat accumulation. These results were accompanied by an increase in IKKɛ phosphorylation and lipogenesis-related proteins such as cluster of differentiation 36 (CD36) and fatty acid synthase (FAS) in the liver of obese mice. In contrast, exercised mice showed lower body weight and adiposity evolution throughout the experiment. In addition, resistance exercise suppressed the effects of the high-fat diet by reducing IKKɛ phosphorylation and hepatic fat content. In conclusion, resistance exercise training improves hepatic fat metabolism and glycaemic homeostasis, which are, at least in part, linked to the anti-inflammatory effect of reduced IKKɛ phosphorylation in the liver of obese mice.

Regulation of Immune Cells by microRNAs and microRNA-Based Cancer Immunotherapy.

MicroRNAs (miRNAs) are small (~21 nucleotides) endogenous noncoding RNA molecules involved in the posttranscriptional regulation of gene expression. Modulation of gene expression by miRNAs occurs via base-pairing of the specific miRNA primary sequence to its corresponding target messenger RNA, which can be located either in the 3' untranslated region or within the coding sequence. This pairing can lead to either translational repression or cleavage of the mRNA, resulting in reduced levels of the target protein. MiRNAs are involved in mediating and controlling several interactions between immune and cancer cells and are also important regulators of immune responses. Increasing interest has focused on elucidating the role of miRNAs in the regulation of anticancer immune responses and how this could affect the efficacy of different cancer therapeutics. Indeed, immune responses have both pro- and anti-oncogenic effects, and functional interactions between immune and cancer cells in the tumor microenvironment are crucial in determining the course of cancer progression. Thus, understanding the role of miRNAs in controlling cancer immunity is important for revealing mechanisms that could be modulated to enhance the success of immunotherapy for patients with cancer. In this chapter, we discuss the involvement of miRNAs in the regulation of immune cells and potential therapeutic approaches in which miRNAs are used for cancer immunotherapy.

Long non-coding RNAs in cancer: Another layer of complexity.

We review the most well characterized long non-coding RNAs (lncRNAs) with important roles in hallmarks of cancer, additionally including lncRNAs with a higher potential for clinical application. LncRNAs are transcripts larger than 200 nucleotides in length that do not appear to have protein-coding potential, although some of those may produce small functional peptides. These transcripts have attracted significant attention from researchers as a result of their role in genetic regulation, including epigenetic, transcriptional and post-transcriptional regulation, being involved in numerous biological processes, as well as being associated with multifactorial diseases, including tumorigenesis. The hallmarks of cancer include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis and activating invasion/metastasis. Additionally, genome instability, inflammation, reprogramming of energy metabolism and evading immune destruction and lncRNAs are implicated in all hallmarks of cancer. Based on the great number of studies describing lncRNAs associated with diverse aspects of most tumor types, lncRNAs have essential roles in potentially all biological features of cancer cells and show great utility as diagnostic and prognostic markers, as exemplified by PCA3 lncRNA detection in prostate cancer diagnosis.

Lnc-uc.147 Is Associated with Disease Stage of Liver, Gastric, and Renal Cancer.

Lnc-uc.147, a long non-coding RNA derived from a transcribed ultraconserved region (T-UCR), was previously evidenced in breast cancer. However, the role of this region in other tumor types was not previously investigated. The present study aimed to investigate lnc-uc.147 in different types of cancer, as well as to suggest lnc-uc.147 functional and regulation aspects. From solid tumor datasets analysis of The Cancer Genome Atlas (TCGA), deregulated lnc-uc.147 expression was associated with the histologic grade of hepatocellular carcinoma, and with the tumor stage of clear cell renal and gastric adenocarcinoma. Considering the epidemiologic relevance of liver cancer, silencing lnc-uc.147 reduced the viability and clonogenic capacity of HepG2 cell lines. Additionally, we suggest a relation between the transcription factor TEAD4 and lnc-uc.147 in liver and breast cancer cells.

The Potential of NORAD-PUMILIO-RALGAPB Regulatory Axis as a Biomarker in Breast Cancer.

Introduction: Long non-coding RNAs (LncRNA) represent a heterogeneous family of RNAs that have emerged as regulators of various biological processes through their association with proteins in ribonucleoproteins complexes. The dynamic of these interactions can affect cell metabolism, including cancer development. Annually, breast cancer causes thousands of deaths worldwide, and searching for new biomarkers is pivotal for better diagnosis and treatment. Methods: Based on in silico prediction analysis, we focus on LncRNAs that have binding sites for PUMILIO, an RBP family involved in post-transcriptional regulation and associated with cancer progression. We compared the expression levels of these LncRNAs in breast cancer and non-tumor samples from the TCGA database. We analyzed the impact of overall and disease-free survival associated with the expression of the LncRNAs and co-expressed genes and targets of PUMILIO proteins. Results: Our results found NORAD as the most relevant LncRNA with a PUMILIO binding site in breast cancer, differently expressed between Luminal A and Basal subtypes. Additionally, NORAD was co-expressed in a Basal-like subtype (0.55) with the RALGAPB gene, a target gene of PUMILIO related to chromosome stability during cell division. Conclusion: These data suggest that this molecular axis may provide insights for developing novel therapeutic strategies for breast cancer.

Transcribed Ultraconserved Regions Are Associated with Clinicopathological Features in Breast Cancer.

Ultraconserved regions (UCRs) are 481 genome segments, with length longer than 200 bp, that are 100% conserved among humans, mice, and rats. The majority of UCRs are transcriptionally active (T-UCRs) as many of them produce non-coding RNAs. In a previous study, we evaluated the expression level of T-UCRs in breast cancer (BC) patients and found that 63% of transcripts correlated with some clinical and/or molecular parameter of BC. In this study, we delved into the expression levels of 12 T-UCRs and correlated them with clinicopathological parameters, immunohistochemical markers, and overall survival in two breast cancer cohorts: TCGA and Brazilian patients. We found that uc.268 is more expressed in TCGA patients under 40 years of age, associated with progesterone receptor (PR) and estrogen receptor (ER), and its high expression is found in luminal A. Lower uc.84 and uc.376 were respectively observed in metastatic and stage IV tumors associated with good prognostic in luminal B. Moreover, uc.84 was only related to the HER2+, while uc.376 was related to ER+ and PR+, and HER2+. A panel composed of uc.147, uc.271, and uc.427 distinguished luminal A from triple negative patients with an AUC of 0.9531 (sensitivity 92.19% and specificity 86.76%). These results highlight the potential role of T-UCRs in BC and provide insights into the potential application of T-UCRs as biomarkers.

Characterization of the Interaction Between SARS-CoV-2 Membrane Protein (M) and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target.

SARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M-PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and PLA (Proximity Ligation Assay). In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase in PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

Altered cancer metabolism in mechanisms of immunotherapy resistance.

Many metabolic alterations, including the Warburg effect, occur in cancer cells that influence the tumor microenvironment, including switching to glycolysis from oxidative phosphorylation, using opportunistic modes of nutrient acquisition, and increasing lipid biosynthesis. The altered metabolic landscape of the tumor microenvironment can suppress the infiltration of immune cells and other functions of antitumor immunity through the production of immune-suppressive metabolites. Metabolic dysregulation in cancer cells further affects the expression of cell surface markers, which interferes with immune surveillance. Immune checkpoint therapies have revolutionized the standard of care for some patients with cancer, but disease in many others is resistant to immunotherapy. Specific metabolic pathways involved in immunotherapy resistance include PI3K-Akt-mTOR, hypoxia-inducible factor (HIF), adenosine, JAK/STAT, and Wnt/Beta-catenin. Depletion of essential amino acids such as glutamine and tryptophan and production of metabolites like kynurenine in the tumor microenvironment also blunt immune cell function. Targeted therapies against metabolic checkpoints could work in synergy with immune checkpoint therapy. This combined strategy could be refined by profiling patients' mutation status before treatment and identifying the optimal sequencing of therapies. This personalized combinatorial approach, which has yet to be explored, may well pave the way for overcoming resistance to immunotherapy.

The anti-inflammatory and pro-resolution effects of aspirin-triggered RvD1 (AT-RvD1) on peripheral blood mononuclear cells from patients with severe asthma.

Asthma is an inflammatory disease that is characterized by a predominance of eosinophils and/or neutrophils in the airways. In the resolution of inflammation, lipid mediators such as resolvin D1 (RvD1) and its epimer aspirin-triggered RvD1 (AT-RvD1) are produced and demonstrate anti-inflammatory and pro-resolution effects. In experimental models such as airway allergic inflammation induced by ovalbumin in mice, RvD1 and AT-RvD1 alleviate some of the most important phenotypes of asthma. Here, we demonstrated the effects of AT-RvD1 on peripheral blood mononuclear cells (PBMCs) from healthy individuals and patients with severe asthma stimulated with lipopolysaccharide (LPS) or Dermatophagoides pteronyssinus (DM). AT-RvD1 (100nM) reduced the concentration of TNF-α in PBMCs from healthy individuals and patients with severe asthma stimulated with LPS or DM. In addition, AT-RvD1 lowered the production of IL-10 only in PBMCs from patients with severe asthma stimulated with LPS. These effects were associated in part with decreasing NF-κB activation. Moreover, AT-RvD1 significantly increased phagocytosis of apoptotic neutrophils by monocytes from patients with severe asthma. In conclusion, AT-RvD1 demonstrated both anti-inflammatory and pro-resolution effects in PBMCs from patients with severe asthma and could become in the future an alternative treatment for asthma.