Chemotherapy induces ACE2 expression in breast cancer via the ROS-AKT-HIF-1α signaling pathway: a potential prognostic marker for breast cancer patients receiving chemotherapy.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin-angiotensin system and a well-known functional receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells. The COVID-19 pandemic has brought ACE2 into the spotlight, and ACE2 expression in tumors and its relationship with SARS-COV-2 infection and prognosis of cancer patients have received extensive attention. However, the association between ACE2 expression and tumor therapy and prognosis, especially in breast cancer, remains ambiguous and requires further investigation. We have previously reported that ACE2 is elevated in drug-resistant breast cancer cells, but the exact function of ACE2 in drug resistance and progression of this malignant disease has not been explored. METHODS: The expression of ACE2 and HIF-1α in parental and drug-resistant breast cancer cells under normoxic and hypoxic conditions was analyzed by Western blot and qRT-PCR methods. The protein levels of ACE2 in plasma samples from breast cancer patients were examined by ELISA. The relationship between ACE2 expression and breast cancer treatment and prognosis was analyzed using clinical specimens and public databases. The reactive oxygen species (ROS) levels in breast cancer cells were measured by using a fluorescent probe. Small interfering RNAs (siRNAs) or lentivirus-mediated shRNA was used to silence ACE2 and HIF-1α expression in cellular models. The effect of ACE2 knockdown on drug resistance in breast cancer was determined by Cell Counting Kit 8 (CCK-8)-based assay, colony formation assay, apoptosis and EdU assay. RESULTS: ACE2 expression is relatively low in breast cancer cells, but increases rapidly and specifically after exposure to anticancer drugs, and remains high after resistance is acquired. Mechanistically, chemotherapeutic agents increase ACE2 expression in breast cancer cells by inducing intracellular ROS production, and increased ROS levels enhance AKT phosphorylation and subsequently increase HIF-1α expression, which in turn upregulates ACE2 expression. Although ACE2 levels in plasma and cancer tissues are lower in breast cancer patients compared with healthy controls, elevated ACE2 in patients after chemotherapy is a predictor of poor treatment response and an unfavorable prognostic factor for survival in breast cancer patients. CONCLUSION: ACE2 is a gene in breast cancer cells that responds rapidly to chemotherapeutic agents through the ROS-AKT-HIF-1α axis. Elevated ACE2 modulates the sensitivity of breast cancer cells to anticancer drugs by optimizing the balance of intracellular ROS. Moreover, increased ACE2 is not only a predictor of poor response to chemotherapy, but is also associated with a worse prognosis in breast cancer patients. Thus, our findings provide novel insights into the spatiotemporal differences in the function of ACE2 in the initiation and progression of breast cancer.

Preliminary investigation of gene expression levels of PAPP-A, STC-2, and HIF-1α in SARS-Cov-2 infected patients.

BACKGROUND: Coronavirus-19 is still considered a pandemic that influences the world. Other molecular alterations should be clearer besides the increasing cytokine storm and pro-inflammatory molecules. Hypoxic conditions that induce HIF-1α lead to stimulate gene expression of STC-2 that targets PAPP-A expression. This study aimed to determine gene expression levels of PAPP-A, STC-2, and HIF-1α in COVID-19 infection. We also aimed to reveal the relationship of these genes with laboratory and clinical data of COVID-19 patients. MATERIALS AND RESULTS: We extracted RNA from peripheral blood samples of COVID-19(+) and COVID-19(-) individuals. The real-time PCR method was used to measure mRNA expression of PAPP-A, STC-2, and HIF-1α. Gene expression analysis was evaluated by the 2-ΔΔCt method. PAPP-A, STC-2, and HIF-1α mRNA expressions of severe patients were higher than healthy individuals (p = 0.0451, p = 0.4466, p < 0.0001, respectively). Correlation analysis of gene expression patterns of severe patients demonstrated a positive correlation between PAPP-A and STC-2 (p < 0.0001, r = 0.8638). CONCLUSION: This is the first study that investigates the relation of PAPP-A, STC-2, and HIF-1α gene expression in patients with COVID-19 infection. Besides the routine laboratory findings, PAPP-A, STC-2, and HIF-1α mRNA expressions may be considered to patients' prognosis as a sign of increased cytokines and pro-inflammatory molecules.

Formation of Neutrophil Extracellular Traps by Reduction of Cellular Cholesterol Is Independent of Oxygen and HIF-1α.

Formation of neutrophil extracellular traps (NETs) is a two-faced innate host defense mechanism, which, on the one hand, can counteract microbial infections, but on the other hand, can contribute to massive detrimental effects on the host. Cholesterol depletion from the cellular membrane by Methyl-ß-cyclodextrin (MßCD) is known as one of the processes initiating NET formation. Since neutrophils mainly act in an inflammatory environment with decreased, so-called hypoxic, oxygen conditions, we aimed to study the effect of oxygen and the oxygen stress regulator hypoxia-inducible factor (HIF)-1α on cholesterol-dependent NET formation. Thus, murine bone marrow-derived neutrophils from wild-type and HIF-knockout mice or human neutrophils were stimulated with MßCD under normoxic (21% O2) compared to hypoxic (1% O2) conditions, and the formation of NETs were studied by immunofluorescence microscopy. We found significantly induced NET formation after treatment with MßCD in murine neutrophils derived from wild-type as well as HIF-1α KO mice at both hypoxic (1% O2) as well as normoxic (21% O2) conditions. Similar observations were made in freshly isolated human neutrophils after stimulation with MßCD or statins, which block the HMG-CoA reductase as the key enzyme in the cholesterol metabolism. HPLC was used to confirm the reduction of cholesterol in treated neutrophils. In summary, we were able to show that NET formation via MßCD or statin-treatment is oxygen and HIF-1α independent.

Adhatoda Vasica attenuates inflammatory and hypoxic responses in preclinical mouse models: potential for repurposing in COVID-19-like conditions.

BACKGROUND: COVID-19 pneumonia has been associated with severe acute hypoxia, sepsis-like states, thrombosis and chronic sequelae including persisting hypoxia and fibrosis. The molecular hypoxia response pathway has been associated with such pathologies and our recent observations on anti-hypoxic and anti-inflammatory effects of whole aqueous extract of Adhatoda Vasica (AV) prompted us to explore its effects on relevant preclinical mouse models. METHODS: In this study, we tested the effect of whole aqueous extract of AV, in murine models of bleomycin induced pulmonary fibrosis, Cecum Ligation and Puncture (CLP) induced sepsis, and siRNA induced hypoxia-thrombosis phenotype. The effect on lung of AV treated naïve mice was also studied at transcriptome level. We also determined if the extract may have any effect on SARS-CoV2 replication. RESULTS: Oral administration AV extract attenuates increased airway inflammation, levels of transforming growth factor-ß1 (TGF-ß1), IL-6, HIF-1α and improves the overall survival rates of mice in the models of pulmonary fibrosis and sepsis and rescues the siRNA induced inflammation and associated blood coagulation phenotypes in mice. We observed downregulation of hypoxia, inflammation, TGF-ß1, and angiogenesis genes and upregulation of adaptive immunity-related genes in the lung transcriptome. AV treatment also reduced the viral load in Vero cells infected with SARS-CoV2. CONCLUSION: Our results provide a scientific rationale for this ayurvedic herbal medicine in ameliorating the hypoxia-hyperinflammation features and highlights the repurposing potential of AV in COVID-19-like conditions.

Increased expression of hypoxia-induced factor 1α mRNA and its related genes in myeloid blood cells from critically ill COVID-19 patients.

BACKGROUND: COVID-19 counts 46 million people infected and killed more than 1.2 million. Hypoxaemia is one of the main clinical manifestations, especially in severe cases. HIF1α is a master transcription factor involved in the cellular response to oxygen levels. The immunopathogenesis of this severe form of COVID-19 is poorly understood. METHODS: We performed scRNAseq from leukocytes from five critically ill COVID-19 patients and characterized the expression of hypoxia-inducible factor1α and its transcriptionally regulated genes. Also performed metanalysis from the publicly available RNAseq data from COVID-19 bronchoalveolar cells. RESULTS: Critically-ill COVID-19 patients show a shift towards an immature myeloid profile in peripheral blood cells, including band neutrophils, immature monocytes, metamyelocytes, monocyte-macrophages, monocytoid precursors, and promyelocytes-myelocytes, together with mature monocytes and segmented neutrophils. May be the result of a physiological response known as emergency myelopoiesis. These cellular subsets and bronchoalveolar cells express HIF1α and their transcriptional targets related to inflammation (CXCL8, CXCR1, CXCR2, and CXCR4); virus sensing, (TLR2 and TLR4); and metabolism (SLC2A3, PFKFB3, PGK1, GAPDH and SOD2). CONCLUSIONS: The up-regulation and participation of HIF1α in events such as inflammation, immunometabolism, and TLR make it a potential molecular marker for COVID-19 severity and, interestingly, could represent a potential target for molecular therapy. Key messages Critically ill COVID-19 patients show emergency myelopoiesis. HIF1α and its transcriptionally regulated genes are expressed in immature myeloid cells which could serve as molecular targets. HIF1α and its transcriptionally regulated genes is also expressed in lung cells from critically ill COVID-19 patients which may partially explain the hypoxia related events.

Clotting disorder in severe acute respiratory syndrome coronavirus 2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human respiratory viral infection that has rapidly progressed into a pandemic, causing significant morbidity and mortality. Blood clotting disorders and acute respiratory failure have surfaced as the major complications among the severe cases of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection. Remarkably, more than 70% of deaths related to COVID-19 are attributed to clotting-associated complications such as pulmonary embolism, strokes and multi-organ failure. These vascular complications have been confirmed by autopsy. This study summarizes the current understanding and explains the possible mechanisms of the blood clotting disorder, emphasizing the role of (1) hypoxia-related activation of coagulation factors like tissue factor, a significant player in triggering coagulation cascade, (2) cytokine storm and activation of neutrophils and the release of neutrophil extracellular traps and (3) immobility and ICU related risk factors.