Notch4 signaling limits regulatory T-cell-mediated tissue repair and promotes severe lung inflammation in viral infections.

A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections.

Mutations in the estrogen receptor alpha hormone binding domain promote stem cell phenotype through notch activation in breast cancer cell lines.

The detection of recurrent mutations affecting the hormone binding domain (HBD) of estrogen receptor alpha (ERα/ESR1) in endocrine therapy-resistant and metastatic breast cancers has prompted interest in functional characterization of these genetic alterations. Here, we explored the role of HBD-ESR1 mutations in influencing the behavior of breast cancer stem cells (BCSCs), using various BC cell lines stably expressing wild-type or mutant (Y537 N, Y537S, D538G) ERα. Compared to WT-ERα clones, mutant cells showed increased CD44+/CD24- ratio, mRNA levels of stemness genes, Mammosphere Forming Efficiency (MFE), Self-Renewal and migratory capabilities. Mutant clones exhibited high expression of NOTCH receptors/ligands/target genes and blockade of NOTCH signaling reduced MFE and migratory potential. Mutant BCSC activity was dependent on ERα phosphorylation at serine 118, since its inhibition decreased MFE and NOTCH4 activation only in mutant cells. Collectively, we demonstrate that the expression of HBD-ESR1 mutations may drive BC cells to acquire stem cell traits through ER/NOTCH4 interplay. We propose the early detection of HBD-ESR1 mutations as a challenge in precision medicine strategy, suggesting the development of tailored-approaches (i.e. NOTCH inhibitors) to prevent disease development and metastatic spread in BC mutant-positive patients.

A Jagged 1-Notch 4 molecular switch mediates airway inflammation induced by ultrafine particles.

BACKGROUND: Exposure to traffic-related particulate matter promotes asthma and allergic diseases. However, the precise cellular and molecular mechanisms by which particulate matter exposure acts to mediate these effects remain unclear. OBJECTIVE: We sought to elucidate the cellular targets and signaling pathways critical for augmentation of allergic airway inflammation induced by ambient ultrafine particles (UFP). METHODS: We used in vitro cell-culture assays with lung-derived antigen-presenting cells and allergen-specific T cells and in vivo mouse models of allergic airway inflammation with myeloid lineage-specific gene deletions, cellular reconstitution approaches, and antibody inhibition studies. RESULTS: We identified lung alveolar macrophages (AM) as the key cellular target of UFP in promoting airway inflammation. Aryl hydrocarbon receptor-dependent induction of Jagged 1 (Jag1) expression in AM was necessary and sufficient for augmentation of allergic airway inflammation by UFP. UFP promoted TH2 and TH17 cell differentiation of allergen-specific T cells in a Jag1- and Notch 4-dependent manner. Treatment of mice with an anti-Notch 4 antibody abrogated exacerbation of allergic airway inflammation induced by UFP. CONCLUSION: UFP exacerbate allergic airway inflammation by promoting a Jag1-Notch 4-dependent interaction between AM and allergen-specific T cells, leading to augmented TH cell differentiation.

Notch4 inhibition suppresses invasion and vasculogenic mimicry formation of hepatocellular carcinoma cells.

Vasculogenic mimicry (VM) is a process by which aggressive tumor cells generate non-endothelial cell-lined channels in malignant tumors including hepatocellular carcinoma (HCC). It has provided new insights into tumor behavior and has surfaced as a potential target for drug therapy. The molecular events underlying the process of VM formation are still poorly understood. In this study, we attempted to elucidate the relationship between Notch4 and VM formation in HCC. An effective siRNA lentiviral vector targeting Notch4 was constructed and transfected into Bel7402, a HCC cell line. VM networks were observed with a microscope in a 3 dimensional cell culture system. Cell migration and invasion were evaluated using wound healing and transwell assays. Matrix metalloproteinases (MMPs) activity was detected by gelatin zymography. Furthermore, the role of Notch4 inhibition in Bel7402 cells in vivo was examined in subcutaneous xenograft tumor model of mice. The results showed that downregulation of Notch4 destroyed VM network formation and inhibited migration and invasion of tumor cells in vitro (P<0.05). In vivo, tumor growth was also inhibited in subcutaneous xenograft model (P<0.05). The potential mechanisms might be related with down-regulation of MT1-MMP, MMP-2, MMP-9 expression and inhibition of the activation of MMP2 and MMP9. These results indicated that Notch4 may play an important role in VM formation and tumor invasion in HCC. Related molecular pathways may be used as novel therapeutic targets for HCC antiangiogenesis therapy.