Hypoxia Dynamically Regulates DBC1 Ubiquitination and Stability by SIAH2 and OTUD5 in Breast Cancer Progression

DBC1 has been characterized as a key regulator of physiological and pathophysiological activities, such as DNA damage, senescence and tumorigenesis. However, the mechanism by which the functional stability of DBC1 is regulated has yet to be elucidated. Here, we report that the ubiquitination-mediated degradation of DBC1 is dynamically regulated by the E3 ubiquitin ligase SIAH2 and deubiquitinase OTUD5 under hypoxic stress. Mechanistically, hypoxia promoted the competitive binding of SIAH2 with OTUD5 to DBC1, resulting in the ubiquitination and subsequent degradation of DBC1 through the ubiquitin-proteasome pathway. Siah2 knockout inhibited tumor cell proliferation and migration, which could be rescued by double knockout of Siah2/DBC1. Human tissue microarray analysis further revealed that the SIAH2/DBC1 axis was responsible for tumor progression under hypoxic stress. These findings define a key role of the hypoxia-mediated SIAH2-DBC1 pathway in the progression of human breast cancer and provide novel insights into the metastatic mechanism of breast cancer.

Transcriptome Analysis of Gills Provides Insights Into Translation Changes Under Hypoxic Stress and Reoxygenation in Golden Pompano, Trachinotus ovatus (Linnaeus 1758)

Golden pompano (Trachinotus ovatus) is one of the most economically critical marine fish in South China. Low oxygen stress has resulted in substantial economic losses to the aquaculture of T. ovatus. However, the molecular responses of fish gills to hypoxia challenge remain unclear. To understand the mechanism underlying adaption to hypoxia, we analyzed the transcriptome of T. ovatus gills in response to hypoxic stress in the normal oxygen group, hypoxic group, and hypoxia treatment after oxygen recovery group. This study found that hypoxia for 8 h was the critical time of hypoxic stress and corresponded to the largest number of differentially expressed genes. After hypoxic stress, genes for chemokines, chemokine receptors, interleukins, complement factors, and other cytokines were significantly downregulated, which may be why fish are vulnerable to pathogen infection in a hypoxic environment. According to a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, many downregulated genes were significantly enriched in the steroid biosynthesis, focal adhesion, and the extracellular matrix (ECM)-receptor interaction signal pathways, which affected cell signal transduction, adhesion, and apoptosis. Compared with the hypoxic group, the amounts of upregulated genes related to phagocytosis and protein degradation were upregulated in the dissolved oxygen recovery group. These results indicated that after the recovery of dissolved oxygen, the fish body repaired the stress-induced damage by rapidly removing misfolded proteins. These findings provide a better understanding of the hypoxia response mechanism of fish and represent a useful resource for the genetic breeding of T. ovatus.

Kynurenine Metabolism Lifespan Extension Mediated by Oxidative Stress Response and Hypoxic Response in C. elegans

Aging is characterized by a progressive decline in the normal physiological functions of an organism, ultimately leading to mortality. Metabolic changes throughout the aging process disrupt the balance and homeostasis of the cell. The kynurenine metabolic pathway is the sole de novo biosynthetic pathway for producing NAD+ from ingested tryptophan. Altered kynurenine pathway activity is associated with both aging and a variety of age-associated diseases, and kynurenine-based interventions can extend lifespan in Caenorhabditis elegans. Our laboratory recently demonstrated knockdown of the kynurenine pathway enzymes kynureninase (KYNU) or 3-hydroxyanthranilic acid dioxygenase (HAAO) increases lifespan by 20-30% in C elegans. However, the mechanism of how these interventions may modulate response against different stressors during the aging process has yet to be explored. Fluorescent reporter strains show the stress-responsive transcription factors skn-1 (ortholog of NRF2/NFE2L2; oxidative stress response) and hif-1 (ortholog of HIF1A; hypoxic stress response) to be highly upregulated when the kynurenine pathway is inhibited. We also demonstrated the increase expression of gst-4 and gcs-1 (transcriptional targets skn-1), which are involved in production of the antioxidant glutathione (GSH), as well as upregulation of cysl-2 (transcriptional target of hif-1), a regulator of cysteine biosynthesis from serine. We hypothesize that lifespan extension resulting from kynurenine pathway inhibition is mediated, at least in part, by upregulation of these transcription factors, providing elevated defense against oxidative stress and hypoxic stress.

EpoR stimulates rapid cycling and larger red cells during mouse and human erythropoiesis

The erythroid terminal differentiation program couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. Here we use Epor−/− mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. We find that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. EpoR-regulation of cell size is independent of established red cell size regulation by iron. High erythropoietin (Epo) increases red cell size in wild-type mice and in human volunteers. The increase in mean corpuscular volume (MCV) outlasts the duration of Epo treatment and is not the result of increased reticulocyte number. Our work shows that EpoR signaling alters the relationship between cycling and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress.

TAMI-25. UPREGULATION OF CREATINE METABOLISM BY MYELOID CELLS RESULTS IN GLIOBLASTOMA PROGRESSION

Malignant brain tumors are uniquely immunosuppressive, with a predominant infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs) and a deficit in T-cells unrivaled to any other tumor. This unique tumor microenvironment (TME) promotes resistance to both conventional and immune therapies for this disease. The underlying mechanisms by which TAMCs promote glioblastoma (GBM) progression are not fully understood. We found that TAMCs specifically upregulate de-novo creatine metabolism within GBM using unbiased genetic and metabolic screening. This metabolic phenotype was confirmed in human GBM patients by comparing peripheral versus tumor-infiltrating myeloid cells. Examination of de-novo creatine generation using Carbon13 arginine flux revealed that TAMCs, but not tumor-infiltrating CD8+ T-cells, can produce creatine. Furthermore, we demonstrate that TAMCs actively secrete de-novo generated creatine into cell cultures. Examination of the single-cell microenvironment of GBM revealed that malignant cells preferentially express the creatine transporter, indicating that TAMC-derived creatine is taken up by GBM. Notably, SLC6A8 is directly upregulated in the context of hypoxia and suggests that creatine uptake is a mechanism to promote survival under hypoxic stress. Indeed, exogenous creatine supplementation promoted both the migration and survival of multiple glioblastoma cell lines in-vitro. Utilizing an established inhibitor of creatine metabolism, β-Guanidinopropionic acid (β -GPA), we found that β -GPA blocks both the migration and survival of glioma cells under hypoxic stress. Lastly, β -GPA also inhibited creatine secretion by TAMCs, showing that creatine blockade can also influence TAMC metabolic phenotype. In the future, we will examine the importance of creatine metabolism on both immune suppression and tumor progression in-vivo. This work provides novel insights into the role of creatine metabolism in GBM and identifies a unique therapeutic avenue for this devastating disease.

Modeling Hypoxic Stress In Vitro Using Human Embryonic Stem Cells Derived Cardiomyocytes Matured by FGF4 and Ascorbic Acid Treatment

Mature cardiomyocytes (CMs) obtained from human pluripotent stem cells (hPSCs) have been required for more accurate in vitro modeling of adult-onset cardiac disease and drug discovery. Here, we found that FGF4 and ascorbic acid (AA) induce differentiation of BG01 human embryonic stem cell–cardiogenic mesoderm cells (hESC-CMCs) into mature and ventricular CMs. Co-treatment of BG01 hESC-CMCs with FGF4+AA synergistically induced differentiation into mature and ventricular CMs. FGF4+AA-treated BG01 hESC-CMs robustly released acute myocardial infarction (AMI) biomarkers (cTnI, CK-MB, and myoglobin) into culture medium in response to hypoxic injury. Hypoxia-responsive genes and potential cardiac biomarkers proved in the diagnosis and prognosis of coronary artery diseases were induced in FGF4+AA-treated BG01 hESC-CMs in response to hypoxia based on transcriptome analyses. This study demonstrates that it is feasible to model hypoxic stress in vitro using hESC-CMs matured by soluble factors.

Menstruation Dysregulation and Endometriosis Development

Endometriosis is a common gynecological condition characterized by the growth of endometrial-like tissue outside of the uterus which may cause symptoms such as chronic pelvic pain or subfertility. Several surgical and medical therapies are available to manage symptoms, but a cure has yet to be determined which can be attributed to the incomplete understanding of disease pathogenesis. Sampson's theory of retrograde menstruation is a widely accepted theory describing how shed endometrial tissue can enter the peritoneal cavity, but other factors are likely at play to facilitate the establishment of endometriosis lesions. This review summarizes literature that has explored how dysregulation of menstruation can contribute to the pathogenesis of endometriosis such as dysregulation of inflammatory mediators, aberrant endometrial matrix metalloproteinase expression, hypoxic stress, and reduced apoptosis. Overall, many of these factors have overlapping pathways which can prolong the survival of shed endometrial debris, increase tissue migration, and facilitate implantation of endometrial tissue at ectopic sites. Moreover, some of these changes are also implicated in abnormal uterine bleeding and endometrial diseases. More research is needed to better understand the underlying mechanisms driving dysregulation of menstruation in endometriosis specifically and identifying specific pathways could introduce new treatment targets. Analyzing menstrual fluid from women with endometriosis for inflammatory markers and other biomarkers may also be beneficial for earlier diagnosis and disease staging.

H9c2 Cardiomyocytes under Hypoxic Stress: Biological Effects Mediated by Sentinel Downstream Targets

The association between diabetes and cardiovascular diseases is well known. Related diabetes macro- and microangiopathies frequently induce hypoxia and consequently energy failure to satisfy the jeopardized myocardium basal needs. Additionally, it is widely accepted that diabetes impairs endothelial nitric oxide synthase (eNOS) activity, resulting in diminished nitric oxide (NO) bioavailability and consequent endothelial cell dysfunction. In this study, we analyzed the embryonic heart-derived H9c2 cell response to hypoxic stress after administration of a high glucose concentration to reproduce a condition often observed in diabetes. We observed that 24 h hypoxia exposure of H9c2 cells reduced cell viability compared to cells grown in normoxic conditions. Cytotoxicity and early apoptosis were increased after exposure to high glucose administration. In addition, hypoxia induced a RhoA upregulation and a Bcl-2 downregulation and lowered the ERK activation observed in normoxia at both glucose concentrations. Furthermore, a significant cell proliferation rate increases after the 1400 W iNOS inhibitor administration was observed. Again, hypoxia increased the expression level of myogenin, a marker of skeletal muscle cell differentiation. The cardiomyocyte gene expression profiles and morphology changes observed in response to pathological stimuli, as hypoxia, could lead to improper ventricular remodeling responsible for heart failure. Therefore, understanding cell signaling events that regulate cardiac response to hypoxia could be useful for the discovery of novel therapeutic approaches able to prevent heart diseases.