Mutation of the Conserved Threonine 8 within the Human ARF Tumour Suppressor Protein Regulates Autophagy

Background: The ARF tumour suppressor plays a well-established role as a tumour suppressor, halting cell growth by both p53-dependent and independent pathways in several cellular stress response circuits. However, data collected in recent years challenged the traditional role of this protein as a tumour suppressor. Cancer cells expressing high ARF levels showed that its expression, far from being dispensable, is required to guarantee tumour cell survival. In particular, ARF can promote autophagy, a self-digestion pathway that helps cells cope with stressful growth conditions arising during both physiological and pathological processes. Methods: We previously showed that ARF is regulated through the activation of the protein kinase C (PKC)-dependent pathway and that an ARF phospho-mimetic mutant on the threonine residue 8, ARF-T8D, sustains cell proliferation in HeLa cells. We now explored the role of ARF phosphorylation in both basal and starvation-induced autophagy by analysing autophagic flux in cells transfected with either WT and ARF phosphorylation mutants by immunoblot and immunofluorescence. Results: Here, we show that endogenous ARF expression in HeLa cells is required for starvation-induced autophagy. Further, we provide evidence that the hyper-expression of ARF-T8D appears to inhibit autophagy in both HeLa and lung cancer cells H1299. This effect is due to the cells’ inability to elicit autophagosomes formation upon T8D expression. Conclusions: Our results lead to the hypothesis that ARF phosphorylation could be a mechanism through which the protein promotes or counteracts autophagy. Several observations underline how autophagy could serve a dual role in cancer progression, either protecting healthy cells from damage or aiding cancerous cells to survive. Our results indicate that ARF phosphorylation controls protein’s ability to promote or counteract autophagy, providing evidence of the dual role played by ARF in cancer progression.

PVT1, a YAP1 dependent stress responsive lncRNA drives ovarian cancer metastasis and chemoresistance

Metastatic growth of ovarian cancer cells into the peritoneal cavity requires adaptation to various cellular stress factors to facilitate cell survival and growth. Here we demonstrate the role of PVT1, one such stress induced long non-coding RNA, in ovarian cancer growth and metastasis. PVT1 is an amplified and overexpressed lncRNA in ovarian cancer with strong predictive value for survival and response to targeted therapeutics. We find that expression of PVT1 is regulated by ovarian tumor cells in response to cellular stress, particularly loss of cell-cell contacts and changes in matrix rigidity occurring in a YAP1 dependent manner. Induction of PVT1 promotes tumor cell survival, growth, and migration. Conversely, reducing PVT1 levels robustly abrogates metastatic behavior and tumor cell dissemination in cell lines and syngeneic transplantation models in vivo. We find that reducing PVT1 causes widespread transcriptome changes leading to alterations in cellular stress response and metabolic pathways including doxorubicin metabolism, which directly impacts chemosensitivity. Together, these findings implicate PVT1 as a promising therapeutic target to suppress metastasis and avoid chemoresistance in ovarian cancer.

Complementary Omics Strategies to Dissect p53 Signaling Networks Under Nutrient Stress

Signaling trough p53 is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.

Long non-coding RNAs (lncRNAs) NEAT1 and MALAT1 are differentially expressed in severe COVID-19 patients: An integrated single-cell analysis

Hyperactive and damaging inflammation is a hallmark of severe rather than mild Coronavirus disease 2019 (COVID-19). To uncover key inflammatory differentiators between severe and mild COVID-19, we applied an unbiased single-cell transcriptomic analysis. We integrated two single-cell RNA-seq datasets with COVID-19 patient samples, one that sequenced bronchoalveolar lavage (BAL) cells and one that sequenced peripheral blood mononuclear cells (PBMCs). The combined cell population was then analyzed with a focus on genes associated with disease severity. The immunomodulatory long non-coding RNAs (lncRNAs) NEAT1 and MALAT1 were highly differentially expressed between mild and severe patients in multiple cell types. Within those same cell types, the concurrent detection of other severity-associated genes involved in cellular stress response and apoptosis regulation suggests that the pro-inflammatory functions of these lncRNAs may foster cell stress and damage. Thus, NEAT1 and MALAT1 are potential components of immune dysregulation in COVID-19 that may provide targets for severity related diagnostic measures or therapy.

ABSENCE OF OXIDATIVE STRESS AND SIRTUINS RECRUITMENT ON CARDIAC TISSUE POST STRESS

Stress has emerged as a factor associated with cardiovascular disease. Catecholamines released during the stress reaction by the sympathetic nerves and the adrenal medulla couple to β1-and β2-adrenoceptors in the cardiomyocytes membrane enhancing heart function in order to attend the organism demand. This might produce excessive reactive oxygen species what may culminate with oxidative stress and progression of several cardiac diseases. Sirtuins have been described as cardioprotective factors and important regulators of the cellular stress response in the heart. The aim of this work is to investigate the putative participation of oxidative stress and sirtuins in the heart of rats submitted to foot shock stress, an experimental model where there is up regulation of β2-adrenoceptors and downregulation of β1-adrenoceptors. The data have shown that in the myocardium of rats submitted to foot shock stress the H2O2 concentration, catalase and superoxide dismutase activity, NAD+/NADH ratio, as well as the protein expression of sirtuins 1 and 3 were not altered. Pharmacological blockade of the β2-adrenoceptors by ICI118,551, did not modify this scenario. It is concluded that foot shock stress does not cause disruptions in oxidative stress or redox state processes in the myocardium, and consequently, sirtuins are not recruited to stress response.

Chemical Characterization, Antitumor, and Immune-Enhancing Activities of Polysaccharide from Sargassum pallidum

Searching for natural products with antitumor and immune-enhancing activities is an important aspect of cancer research. Sargassum pallidum is an edible brown alga that has been used in Chinese traditional medicine for the treatment of tumors. However, the purification and application of its active components are still insufficient. In the present study, the polysaccharides from S. pallidum (SPPs) with antitumor and immune-enhancing activities were isolated and purified, and five polysaccharide fractions (SPP-0.3, SPP-0.5, SPP-0.7, SPP-1, and SPP-2) were obtained. The ratio of total saccharides, monosaccharide composition, and sulfated contents was determined, and their structures were analyzed by Fourier transform infrared spectroscopy. Moreover, bioactivity analysis showed that all five fractions had significant antitumor activity against three types of cancer cells (A549, HepG2, and B16), and can induce cancer cell apoptosis. In addition, the results indicated that SPPs can enhance the proliferation of immune cells and improve the expression levels of serum cytokines (IL-6, IL-1β, iNOS, and TNF-α). SPP-0.7 was identified as the most active fraction and selected for further purification, and its physicochemical properties and antitumor mechanism were further analyzed. Transcriptome sequencing result showed that SPP-0.7 can significantly induce the cell apoptosis, cytokine secretion, and cellular stress response process, and inhibit the normal physiological processes of cancer cells. Overall, SPPs and SPP-0.7 may be suitable for use as potential candidate agents for cancer therapy.

Loss of FVIII Expression by Possible Interlinked Immune and Cellular Stress Response in Hemophilia A Mice Treated with AAV Gene Therapy

Background Hemophilia A is an X-linked genetic disorder caused by a mutation in the gene for factor VIII (FVIII) protein that reduces the ability of blood to clot. Clinical drug trials have shown the potential of adeno-associated virus (AAV) gene therapy as a one-time treatment for hemophilia A that can produce sustained high levels of FVIII. However, a gradual decline in protein levels has been observed in patients after 2-4 years. The hypothesis being tested in the Herzog Lab is that an interlinked immune and cellular stress response could be causing the loss of expression. Methods Two groups of Hemophilia A mice were administered AAV therapy, with one group receiving recurrent doses of Rapamycin. Blood samples were taken at weeks 4, 8, 12 and 14. Mice were euthanized at weeks 4, 8, and 14, and their livers were harvested. qPCR was used to measure AAV copy numbers and FVIII mRNA at 4, 8, and 14 weeks. Cryosections of mice livers from weeks 4, 8, and 14 were stained with antibodies for FVIII protein and CD8. Results qPCR showed roughly half as much AAV copy numbers in the rapamycin group at all time points, and little difference in FVIII mRNA between the groups. There was also a large decrease in AAV copy numbers and FVIII mRNA in both groups between 8 and 14 weeks. Immunohistochemistry showed less CD8 and more FVIII signal in mice treated with rapamycin. Discussion Experiments are currently being performed to investigate the decline in AAV copy numbers and mRNA between weeks 8 and 14. The immunohistochemistry data shows a relationship between increased FVIII protein levels and decreased cellular immune response but does not explain the gradual decline in FVIII. Further investigation into FVIII expression following AAV gene therapy could lead to an effective one-time treatment for hemophilia A.

Environmental Particulate Air Pollution Exposure and the Oxidative Stress Responses: A Brief Review of the Impact on the Organism and Animal Models of Research

Particulate matter (PM) is a mixture of solid particles and liquid droplets found in the air, and it is one of the most harmful air pollutants. When inhaled, it affects the pulmonary system, cardiovascular systems, and other tissues. The size, composition, and deposition of PM, mainly related to fine and ultrafine particulate matter, are factors that determine the harmful effects of exposure to particles. Among the main effects is the inducer of ROS production, and consequently oxidative tissue damage in target organs and other responses, mediated by inflammatory cytokines and cellular stress response. The main pathway through which particles are potent mediators of oxidative stress is the damage caused to DNA and lipid molecules, whereas the pro-inflammatory response involves an immune response against PM, which in turn, it is related to cell stress responses observed by heat shock proteins (HSPs) expression and release. Thus, the ability of an organism to respond to PM inhalation requires anti-oxidative, anti-inflammatory, and cellular stress defenses that can be impaired in susceptible subjects as people with chronic diseases as diabetes and obesity. In this chapter, we discuss the mechanistic aspects of PM effects on health and present some animal research models in particle inhalation studies.

Effect of Combined Stressors on C. elegans Lifespan

Cellular stress is a fundamental component of age-associated disease. Cells experience many forms of stress (oxidative, heavy metal, etc.), and as we age the burden of stress and resulting damage increases while our cells’ ability to deal with the consequences becomes diminished due to dysregulation of cellular stress response pathways. By understanding how cells respond to stress we aim to slow age-associated deterioration and develop treatment targets for age-associated disease. The majority of past work has focused on understanding responses to individual stressors. In contrast, how pathology and stress responses differ in the presence of multiple stressors is relatively unknown; we investigate that here. We cultured worms on agar plates with different combinations of arsenic, copper, and DTT (which create oxidative/proteotoxic, heavy metal, and endoplasmic reticulum (ER) stress, respectively) at doses that result in 20% lifespan reduction individually and measured the effect on lifespan. We found that arsenic/copper and arsenic/DTT combinations created additive lifespan reductions while the copper/DTT combination created an antagonistic lifespan reduction when compared to controls (p<0.05). This antagonistic toxicity suggests an interaction either between the mechanisms of toxicity or the cellular response to copper and DTT. We are now evaluating the impact of copper and DTT individually and in combination on unfolded protein and heavy metal response pathways to understand the underlying mechanism of the interaction. Additionally, we are continuing to screen stressors to identify combinations that cause non-additive (synergistic or antagonistic) toxicity to build a comprehensive model of the genetic stress response network in C. elegans.