Notch Intracellular Domain Research Articles

M6 A-Dependent Modulation via IGF2BP3/MCM5/Notch Axis Promotes Partial EMT and LUAD Metastasis.

The importance of mRNA N6-methyladenosine (m6 A) modification during tumor metastasis is controversial as it plays distinct roles in different biological contexts. Moreover, how cancer cell plasticity is shaped by m6 A modification is interesting but remains uncharacterized. Here, this work shows that m6 A reader insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) is remarkably upregulated in metastatic lung adenocarcinoma (LUAD) and indicates worse prognosis of patients. Interestingly, IGF2BP3 induces partial epithelial-mesenchymal-transition (EMT) and confers LUAD cells plasticity to metastasize through m6 A-dependent overactivation of Notch signaling. Mechanistically, IGF2BP3 recognized m6 A-modified minichromosome maintenance complex component (MCM5) mRNAs to prolong stability of them, subsequently upregulating MCM5 protein, which competitively inhibits SIRT1-mediated deacetylation of Notch1 intracellular domain (NICD1), stabilizes NICD1 protein and contributes to m6 A-dependent IGF2BP3-mediated cellular plasticity. Notably, a tight correlation of the IGF2BP3/MCM5/Notch axis is evidenced in clinical LUAD specimens. Therefore, this study elucidates a critical role of m6 A modification on LUAD cell plasticity in fostering tumor metastasis via the above axis, providing potential targets for metastatic LUAD.

Inhibition of the intracellular domain of Notch1 results in vascular endothelial cell dysfunction in sepsis.

Notch signaling is critical for regulating the function of vascular endothelial cells (ECs). However, the effect of the intracellular domain of Notch1 (NICD) on EC injury in sepsis remains unclear. We established a cell model of vascular endothelial dysfunction and induced sepsis in a mouse model via lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP). Endothelial barrier function and expression of endothelial-related proteins were determined using CCK-8, permeability, flow cytometry, immunoblot, and immunoprecipitation assays. The effect of NICD inhibition or activation on endothelial barrier function was evaluated in vitro. Melatonin was used for NICD activation in sepsis mice. The survival rate, Evans blue dye of organs, vessel relaxation assay, immunohistochemistry, ELISA, immunoblot were used to explore the specific role of melatonin for sepsis induced vascular dysfunction in vivo. We found that LPS, interleukin 6, and serum collected from septic children could inhibit the expression of NICD and its downstream regulator Hes1, which impaired endothelial barrier function and led to EC apoptosis through the AKT pathway. Mechanistically, LPS decreased the stability of NICD by inhibiting the expression of a deubiquitylating enzyme, ubiquitin-specific proteases 8 (USP8). Melatonin, however, upregulated USP8 expression, thus maintaining the stability of NICD and Notch signaling, which ultimately reduced EC injury in our sepsis model and elevated the survival rate of septic mice. We found a previously uncharacterized role of Notch1 in mediating vascular permeability during sepsis, and we showed that inhibition of NICD resulted in vascular EC dysfunction in sepsis, which was reversed by melatonin. Thus, the Notch1 signaling pathway is a potential target for the treatment of sepsis.

Mechanism of Notch3 signaling pathway regulating the differentiation of aortic dissection vascular stem cells into smooth muscle cells

To explore whether the differentiation of vascular stem cells (VSC) into smooth muscle cells (SMC) in aortic dissection (AD) is dysregulated, and to verify the role of Notch3 pathway in this process. Aortic tissues were obtained from AD patients undergoing aortic vascular replacement and heart transplant donors at Department of Cardiovascular Surgery, Guangdong Provincial People's Hospital Affiliated to Southern Medical University. VSC were isolated by enzymatic digestion and c-kit immunomagnetic beads. The cells were divided into normal donor-derived VSC group (Ctrl-VSC group) and AD-derived VSC group (AD-VSC group). The presence of VSC in the aortic adventitia was detected by immunohistochemical staining, and VSC was identified by stem cell function identification kit. The differentiation model of VSC into SMC established in vitro was induced by transforming growth factor-β1 (10 μg/L) for 7 days. They were divided into normal donor VSC-SMC group (Ctrl-VSC-SMC group), AD VSC-SMC group (AD-VSC-SMC group) and AD VSC-SMC+Notch3 inhibitor DAPT group (AD-VSC-SMC+DAPT group,DAPT 20 μmol/L was added during differentiation induction). The expression of contractile marker Calponin 1 (CNN1) in SMC derived from aortic media and VSC were detected by immunofluorescence staining. The protein expressions of contractile markers α-smooth muscle actin (α-SMA), CNN1 as well as Notch3 intracellular domain (NICD3) in SMC derived from aortic media and VSC were detected by Western blotting. Immunohistochemical staining showed there was a population of c-kit-positive VSC in the adventitia of aortic vessels, and VSC from both normal donors and AD patients had the ability to differentiate into adipocytes and chondrocytes. Compared with normal donor vascular tissue, the expressions of SMC markers α-SMA and CNN1 of tunica media contraction in AD were down-regulated (α-SMA/β-actin: 0.40±0.12 vs. 1.00±0.11, CNN1/β-actin: 0.78±0.07 vs. 1.00±0.14, both P < 0.05), while the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 2.22±0.57 vs. 1.00±0.15, P < 0.05). Compared with Ctrl-VSC-SMC group, the expressions of contractile SMC markers α-SMA and CNN1 were down-regulated in AD-VSC-SMC group (α-SMA/β-actin: 0.35±0.13 vs. 1.00±0.20, CNN1/β-actin: 0.78±0.06 vs. 1.00±0.07, both P < 0.05), the protein expression of NICD3 was up-regulated (NICD3/GAPDH: 22.32±1.22 vs. 1.00±0.06, P < 0.01). Compared with AD-VSC-SMC group, the expressions of contractile SMC markers α-SMA, CNN1 were up-regulated in AD-VSC-SMC+DAPT group (α-SMA/β-actin: 1.70±0.07 vs. 1.00±0.15, CNN1/β-actin: 1.62±0.03 vs. 1.00±0.02, both P < 0.05). Dysregulation of VSC differentiation into SMC occurs in AD, while inhibition of Notch3 pathway activation can restore the expression of contractile proteins in VSC-derived SMC in AD.

Glia maturation factor-γ is required for initiation and maintenance of hematopoietic stem and progenitor cells

BackgroundIn vertebrates, hematopoietic stem and progenitor cells (HSPCs) emerge from hemogenic endothelium in the floor of the dorsal aorta and subsequently migrate to secondary niches where they expand and differentiate into committed lineages. Glia maturation factor γ (gmfg) is a key regulator of actin dynamics that was shown to be highly expressed in hematopoietic tissue. Our goal is to investigate the role and mechanism of gmfg in embryonic HSPC development.MethodsIn-depth bioinformatics analysis of our published RNA-seq data identified gmfg as a cogent candidate gene implicated in HSPC development. Loss and gain-of-function strategies were applied to study the biological function of gmfg. Whole-mount in situ hybridization, confocal microscopy, flow cytometry, and western blotting were used to evaluate changes in the number of various hematopoietic cells and expression levels of cell proliferation, cell apoptosis and hematopoietic-related markers. RNA-seq was performed to screen signaling pathways responsible for gmfg deficiency-induced defects in HSPC initiation. The effect of gmfg on YAP sublocalization was assessed in vitro by utilizing HUVEC cell line.ResultsWe took advantage of zebrafish embryos to illustrate that loss of gmfg impaired HSPC initiation and maintenance. In gmfg-deficient embryos, the number of hemogenic endothelium and HSPCs was significantly reduced, with the accompanying decreased number of erythrocytes, myelocytes and lymphocytes. We found that blood flow modulates gmfg expression and gmfg overexpression could partially rescue the reduction of HSPCs in the absence of blood flow. Assays in zebrafish and HUVEC showed that gmfg deficiency suppressed the activity of YAP, a well-established blood flow mediator, by preventing its shuttling from cytoplasm to nucleus. During HSPC initiation, loss of gmfg resulted in Notch inactivation and the induction of Notch intracellular domain could partially restore the HSPC loss in gmfg-deficient embryos.ConclusionsWe conclude that gmfg mediates blood flow-induced HSPC maintenance via regulation of YAP, and contributes to HSPC initiation through the modulation of Notch signaling. Our findings reveal a brand-new aspect of gmfg function and highlight a novel mechanism for embryonic HSPC development.

FAM129A promotes self-renewal and maintains invasive status via stabilizing the Notch intracellular domain in glioma stem cells.

Glioma stem cells (GSCs) are a subpopulation of tumor cells with self-renewal and tumorigenic capabilities in glioblastomas (GBMs). Diffuse infiltration of GSCs facilitates tumor progression and frustrates efforts at effective treatment. Further compounding this situation is the currently limited understanding of what drives GSC invasion. Here we comprehensively evaluated the significance of a novel invasion-related protein, Family with Sequence Similarity 129 Member A (FAM129A), in infiltrative GSCs. Western blotting, immunohistochemistry, and gene expression analysis were used to quantify FAM129A in glioma specimens and cancer datasets. Overexpression and knockdown of FAM129A in glioma stem cells were used to investigate its effects on tumor growth and invasion. RNA-seq, qRT-PCR, western blotting, and co-precipitation assays were used to investigate FAM129A signaling mechanisms. FAM129A is preferentially expressed in invasive frontiers. Targeting FAM129A impairs GSC invasion and self-renewal. Mechanistically, FAM129A acted as a positive regulator of Notch signaling by binding with the Notch1 intracellular domain (NICD1) and preventing its degradation. FAM129A and NICD1 provide a precise indicator for identifying tumor margins and aiding prognosis. Targeting them may provide a significantly therapeutic strategy for GSCs.

The Notch pathway regulates autophagy after hypoxic-ischemic injury and affects synaptic plasticity.

Following neonatal hypoxic-ischemia (HI) injury, it is crucial factor to reconstruct neural circuit and maintain neural network homeostasis for neurological recovery. A dynamic balance between the synthesis and degradation of synaptic protein is required for maintaining synaptic plasticity. Protein degradation is facilitated by autophagy. This study aimed to investigate the regulation of synaptic structural plasticity by the Notch pathway, by assessing changes in Notch pathway activation and their effects on synaptic proteins and autophagy after HI injury. The study involved 48 male newborn Yorkshire piglets, each weighing 1.0-1.5kg and 3days old. They were randomly assigned to two groups: the HI group and the Notch pathway inhibitor + HI group (n = 24 per group). Each group was further divided into six subgroups according to HI duration (n = 4 per group): a control subgroup, and 0-6, 6-12, 12-24, 24-48, and 48-72h subgroups. The expression of Notch pathway-related proteins, including Notch1, Hes1, and Notch intracellular domains, increased following HI injury. The expression of autophagy proteins increased at 0-6h and 6-12h post-HI. The expression of synaptic proteins, such as postsynaptic density protein 95 (PSD95) and synaptophysin, increased 6-12h and 12-24h after HI, respectively. Notably, the increased expression of these proteins was reversed by a Notch pathway inhibitor. Transmission electron microscopy revealed the presence of autophagosome structures in synapses. These findings shed light on the underlying mechanisms of neurological recovery after HI injury and may provide insights into potential therapeutic targets for promoting neural circuit reconstruction and maintaining neural network homeostasis.

BCL6B Contributes to Ocular Vascular Diseases via Notch Signal Silencing

Endothelial cell activation is tightly controlled by the balance between VEGF (vascular endothelial cell growth factor) and Notch signaling pathway. VEGF destabilizes blood vessels and promotes neovascularization, which are common features of sight-threatening ocular vascular disorders. Here, we show that BCL6B (B-cell CLL/lymphoma 6 member B protein), also known as BAZF, ZBTB28, and ZNF62, plays a pivotal role in the development of retinal edema and neovascularization. The pathophysiological physiological role of BCL6B was investigated in cellular and animal models mimicking 2 pathological conditions: retinal vein occlusion and choroidal neovascularization. An in vitro experimental system was used in which human retinal microvascular endothelial cells were supplemented with VEGF. Choroidal neovascularization cynomolgus monkey model was generated to investigate the involvement of BCL6B in the pathogenesis. Mice lacking BCL6B or treated with BCL6B-targeting small-interfering ribose nucleic acid were examined for histological and molecular phenotypes. In retinal endothelial cells, the BCL6B expression level was increased by VEGF. BCL6B-deficient endothelial cells showed Notch signal activation and attenuated cord formation via blockage of the VEGF-VEGFR2 signaling pathway. Optical coherence tomography images showed that choroidal neovascularization lesions were decreased by BCL6B-targeting small-interfering ribose nucleic acid. Although BCL6B mRNA expression was significantly increased in the retina, BCL6B-targeting small-interfering ribose nucleic acid suppressed ocular edema in the neuroretina. The increase in proangiogenic cytokines and breakdown of the inner blood-retinal barrier were abrogated in BCL6B knockout (KO) mice via Notch transcriptional activation by CBF1 (C promotor-binding factor 1) and its activator, the NICD (notch intracellular domain). Immunostaining showed that Müller cell activation, a source of VEGF, was diminished in BCL6B-KO retinas. These data indicate that BCL6B may be a novel therapeutic target for ocular vascular diseases characterized by ocular neovascularization and edema.

Casein kinase 1α is required to maintain murine hypothalamic pro-opiomelanocortin expression

Hypothalamic pro-opiomelanocortin (POMC) neuron development is considered to play an essential role in the development of obesity. However, the underlying mechanisms remain unclear. Casein kinase 1α (CK1α) was expressed in the embryonic mouse hypothalamus at high levels and colocalized with POMC neurons. CK1α deletion in POMC neurons caused weight gain, metabolic defects, and increased food intake. The number of POMC-expressing cells was considerably decreased in Csnk1a1fl/fl;POMCcre (PKO) mice from embryonic day 15.5 to postnatal day 60, while apoptosis of POMC neurons was not affected. Furthermore, unchanged POMC progenitor cells and a decreased POMC phenotype established CK1α function in hypothalamic POMC neuron development. CK1α deletion led to elevated Notch intracellular domain (NICD) protein expression, and NICD inhibition rescued the PKO mouse phenotype. In summary, CK1α is involved in hypothalamic POMC expression via NICD-POMC signaling, deepening our understanding of POMC neuron development and control of systemic metabolic functions.

Notch activation promotes bone metastasis via SPARC inhibition in adenoid cystic carcinoma.

We aimed to investigate bone metastasis induced by Notch signalling pathway dysregulation and to demonstrate that SPARC is a potential therapeutic target in adenoid cystic carcinoma (AdCC) with Notch dysregulation. This retrospective study enrolled 144 AdCC patients. RNA-sequencing and enrichment analyses were performed using 32 AdCC samples. Osteonectin/SPARC and the Notch activation indicator Notch intracellular domain (NICD) were detected using immunohistochemistry. Cell proliferation and migration assays were conducted using stably NICD over-expressing cells. The effect of SPARC on osteoclast differentiation in NICD cells was investigated using western blotting, quantitative reverse transcription PCR, tartrate-resistant acid phosphatase staining and resorption assays. RNA-sequencing analysis showed that genes down-regulated in Notch-mutant AdCCs, such as SPARC, were enriched in ossification and osteoblast differentiation. Most (75/110, 68.2%) Notch1-wild-type AdCCs showed SPARC over-expression, whereas 30 out of 34 (88.2%) Notch1-mutant tumours showed low SPARC expression. SPARC over-expression was then found negatively to be correlated with NICD expression in 144 AdCCs. NICD over-expression promoted cell growth, migration and osteoclast differentiation, which could be partly reversed by exogenous SPARC. Notch activation in AdCC contributes to bone metastasis through SPARC inhibition. The study results suggest that SPARC may represent a prognostic biomarker and potential therapeutic target.

Abstract 2594: Alcohol drinking inhibits NOTCH-PAX9 signaling in esophageal squamous epithelial cells

Abstract Alcohol drinking has been established as a major risk factor for esophageal diseases, such as gastroesophageal reflux disease (GERD) and esophageal squamous cell carcinoma (ESCC). Certain mechanisms of alcohol-induced esophageal injury are supported by experimental data, whereas most are speculative or extrapolated from studies on cancers of other organ sites. Our previous study showed that ethanol exposure inhibited PAX9 expression in human esophageal squamous epithelial cells in vitro and in vivo, and promoted ESCC. In this study, we aimed to investigate the molecular pathways through which alcohol drinking suppresses PAX9 in esophageal squamous epithelial cells. Gene microarray, qRT-PCR, ChIP-PCR, Western blotting, and immunostaining were used to identify and validate inhibition of NOTCH-PAX9 signaling in human esophageal squamous epithelial cells and mouse esophagus after ethanol exposure. Human tissue samples were analyzed to further validate NOTCH inhibition by alcohol drinking. We first demonstrated the inhibition of NOTCH by ethanol exposure in vitro. NOTCH regulated PAX9 expression in esophageal squamous epithelial cells in vitro and in vivo. RBPJ and NOTCH intracellular domain (NICD1) ChIP-PCR confirmed Pax9 as a direct downstream target of NOTCH signaling in mouse esophagus. NOTCH inhibition by alcohol drinking was further validated in mouse esophagus and human tissue samples. In conclusion, ethanol exposure inhibited NOTCH signaling and thus suppressed PAX9 expression in esophageal squamous epithelial cells in vitro and in vivo. Our data support a novel mechanism of alcohol-induced esophageal injury through the inhibition of NOTCH-PAX9 signaling. As a result, chemical NOTCH activators may be used to prevent or treat alcohol-associated esophageal diseases such as GERD and ESCC. Citation Format: Zhaohui Xiong. Alcohol drinking inhibits NOTCH-PAX9 signaling in esophageal squamous epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2594.

Abstract 4962: Discovery of cancer-specific E3 ligase ligands for targeted protein degradation

Abstract F-box/WD repeat-containing protein 7 (FBXW7) is the substrate recognition component of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex which mediates the ubiquitination and subsequent proteasomal degradation of target proteins. FBXW7 recognizes and binds phosphor-degrons within target proteins and capture them to SCF complex for ubiquitination. Know FBXW7 substrates include cyclin-E, DISC1, JUN, MYC, NFE2L1, NOTCH2, MCL1, RICTOR, and NOTCH1 released notch intracellular domain (NICD). FBXW7 is frequently mutated in cancer, results in loss of substrate recognition and binding. R465C is a one of the major FBXW7 mutations and is found in over 70,000 cancer patients worldwide. From a high-throughput screen, we discovered compounds that selectively bind to FBXW7 R465C mutant. PROTACs against various protein targets have been made with these mutant specific ligands. Some of these PROTACs could induce targeted protein degradation in cells carrying FBXW7 R465C mutant but not wild-type FBXW7. Targeted protein degradation with these PROTACs could represent a novel therapeutic approach against FBXW7 R465C mutant cancer. Citation Format: Zhaofu Wang, Lynette Zhang, Zhen Chang, Mengxi Zhao, Guobin Li, Fang Qin, Shichao Ma, Yunfeng Li, Zhongguo Zhang, Xinghao Wang, Ying Kong, Shidi Lou, Sheng Chen, Lanzhen Liu, Shuaijie Xu, Hailong Zhang. Discovery of cancer-specific E3 ligase ligands for targeted protein degradation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4962.

Abstract 4955: Kurarinone, a natural notch signaling inhibitor and modulator of breast cancer stemness

Abstract Aberrant activation of Notch signaling pathway plays an important role in breast cancer development, progression, and stem cell renewal. Activation of Notch signaling is triggered by the interaction of Notch receptors with different ligands expressed on neighboring cells that bind and induce a second cleavage by a membrane-tethered metalloprotease. The remaining membrane-bound fragment is cleaved by γ-secretase (GS) releasing the Notch Intracellular Domain (NICD), which translocate to the nucleus to activate the expression of Notch target genes. Current indirect approaches to inhibit Notch signaling are based on agents that bind to GS substrates. In the search of novel Notch signaling inhibitor(s), we screened a number of plant flavonoids for their binding potential to GS substrate. Our in-silico study identified kurarinone (KU), a lavandulyl flavanone, having ability to bind to GS complex. The KU-GS complex was structurally and energetically stable during the molecular dynamics simulation period. Interestingly, dose and time dependent toxicity of KU was observed in T47D and MDA-MB-231 breast cancer cells at 1-25µM concentration. KU treatment of cells led to inhibition in cyclin D1 expression, arrest of cells in the G1 phase of the cell cycle and subsequently undergoes apoptosis. KU-mediated apoptosis was a result of altered mitochondria membrane potential and increased ROS production. In dual-luciferase Notch promoter assay, KU inhibited GS-mediated NICD production in transfected HEK293 cells. Furthermore, KU treatment decreased the mammosphere formation potential of MCF-7 cells through altered gene expression involved in self-renewal and stemness. KU treated MDA-MB-231 cells and MCF-7 derived mammosphere showed downregulation of Notch signaling through alteration in the expression of downstream genes such as Hey1, Hes1, and E-cadherin at both transcript and protein levels. Taken together, the present study identified KU as a novel natural Notch signaling inhibitor that could provide a novel and safer approach for the treatment of breast cancer through reduction in stemness and self-renewal properties. Citation Format: Prem Prakash Kushwaha, Mohd Shuaib, Kumari Sunita Prajapati, Atul Kumar Singh, Mahesh Kulharia, Shashank Kumar, Sanjay Gupta. Kurarinone, a natural notch signaling inhibitor and modulator of breast cancer stemness. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4955.

NDRG2 regulates the formation of reactive astrocyte-derived progenitor cells via Notch signaling pathway after brain traumatic injury in rats.

In response to traumatic brain injury, a subpopulation of cortical astrocytes is activated, resulting in acquisition of stem cell properties, known as reactive astrocytes-derived progenitor cells (Rad-PCs). However, the underlying mechanisms remain largely unknown during this process. In this study, we examined the role of N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, in Rad-PCs after cortical stab injury in adult rats. Immunohistochemical analysis showed that in the cerebral cortex of normal adult rats, NDRG2 was exclusively expressed in astrocytes. After liu cortical injury, the expression of NDRG2 was significantly elevated around the wound and most cells expressing NDRG2 also expressed GFAP, a reactive astrocyte marker. Importantly, NDRG2-expressing cells were co-labeled with Nestin, a marker for neural stem cells, some of which also expressed cell proliferation marker Ki67. Overexpression of NDRG2 further increased the number of NDRG2/Nestin double-labeling cells around the lesion. In contrast, shRNA knockdown of NDRG2 decreased the number of NDRG2+/Nestin+ cells. Intracerebroventricular administration of stab-injured rats with a Notch antagonist, DAPT, led to a significant decrease in Nestin+/NDRG2+ cells around the injured boundary, but did not affect NDRG2+ cells. Moreover, overexpression or knockdown of NDRG2 led to up- and down-regulation of the expression of Notch intracellular domain NICD and Notch target gene Hes1, respectively. Taken together, these results suggest that NDRG2 may play a role in controlling the formation of Rad-PCs in the cerebral cortex of adult rats following traumatic injury, and that Notch signaling pathway plays a key role in this process.

Computational analysis of natural compounds targeted for NICD of notch pathway in hepatocellular carcinoma

Therapeutic drugs are required to fight against Hepatocellular carcinoma (HCC), which is one of the prominent cancer types and is considered the world's second leading cause of death related to cancer. HCC is caused by chronic usage of alcohol, obesity, hepatitis virus etc. Currently, the chemotherapeutics drugs available possess severe side effects and face multi-drug resistance due to which the mortality rate is high. The normal liver shows lower expression of notch receptors in comparison to HCC patients where overexpression of notch receptors is observed in the patient’s liver. The present study is based on screening natural compound databases using structure-based virtual screening to find compounds with the capability to inhibit the expression of Notch signaling pathway. 7929 compounds from three natural compound databases (NANPDB, EANPDB, and SANCDB) were subjected to LibDock succeeded by ADME and toxicity prediction using Biovia Discovery Studio Suite. To observe the binding system, molecular docking was performed between the natural compounds and the Notch intracellular domain (target molecule). The computational analysis resulted in a novel natural compound i.e., 5-(2′',3′'-epoxy-3′'-methylbutyl)-3-(3′-hydroxy-3′-methyl-1′-acetyloxy-but-2′-yl)indole. In silico ADMET analysis showed that this compound had good absorption levels with minimal toxic effects. In future, this compound can be utilized as a lead molecule for the development of an anti-HCC medication.

Adenovirus-mediated effects of Wnt and Notch signalling pathways on hair cell regeneration in mice

Some genes are delivered to cochleae by adenoviruses to restore partial hearing function. This provides promising prospects for gene therapies for hearing loss from hair cell damage. To study the adenovirus (AD)-mediated effect of the Wnt and Notch signalling pathways on hair cell regeneration in the mouse cochlea, we constructed a β-catenin-adenovirus (β-catenin-AD) to increase the activity of the Wnt signalling pathway and a NICD (intracellular domain of Notch1)-RNAi-adenovirus to decrease the activity of the Notch signalling pathway (NICD-RNAi-AD). Our study indicated that approximately 40% of supporting cells in the cochleae damaged by gentamicin were infected with the adenoviruses. Following the β-catenin-AD-mediated increase in Wnt signalling pathway activity, mitotic regeneration was increased, while direct transdifferentiation was increased after the NICD-RNAi-AD-mediated decrease in Notch signalling pathway activity. The expected synergistic interaction on hair cell regeneration was not obtained after coinfection of β-catenin-AD and NICD-RNAi-AD into the damaged cochleae, which might be due to the low cotransfection efficiency to supporting cells. Our study indicated that it may be possible to develop AD mediated gene therapies for hearing loss that act by regulating the Wnt and Notch signalling pathways.

SUMO specific peptidase 6 regulates the crosstalk between podocytes and glomerular endothelial cells in diabetic kidney disease

There is increasing evidence that the crosstalk between podocytes and glomerular endothelial cells (GECs) exacerbates the progression of diabetic kidney disease (DKD). Here, we investigated the underlying role of SUMO specific peptidase 6 (SENP6) in this crosstalk. In the diabetic mice, SENP6 was decreased in glomerular tissues and its knockdown further exacerbated glomerular filtration barrier injury. In the mouse podocyte cell line MPC5 cells, SENP6 overexpression reversed HG-induced podocyte loss by suppressing the activation of Notch1 signaling. Notch1 intracellular domain (N1ICD) is the active form of Notch1. SENP6 upregulated the ubiquitination of N1ICD by deSUMOylating Notch1, thereby reducing N1ICD and suppressing Notch1 signaling activation in MPC5 cells. Endothelin-1 (EDN1) is a protein produced by podocytes and has been reported to promote GEC dysfunction. The supernatant from HG-treated MPC5 cells induced mitochondrial dysfunction and surface layer injury in GECs, and the supernatant from SENP6-deficient podocytes further exacerbated the above GEC dysfunction, while this trend was reversed by an EDN1 antagonist. The following mechanism study showed that SENP6 deSUMOylated KDM6A (a histone lysine demethylase) and then decreased the binding potency of KDM6A to EDN1. The latter led to the upregulation of H3K27me2 or H3K27me3 of EDN1 and suppressed its expression in podocytes. Taken together, SENP6 suppressed the HG-induced podocyte loss and ameliorated GEC dysfunction caused by crosstalk between podocytes and GECs, and the protective effect of SENP6 on DKD is attributed to its deSUMOylation activity.

KRT5 mutation regulate melanin metabolism through notch signalling pathway between keratinocytes and melanocytes.

Dowling-Degos disease (DDD) is an autosomal dominant hereditary skin disease characterized by acquired reticular hyperpigmentation in flexural sites, and one of its causative genes is KRT5 gene. But the effect of KRT5, expressed only in keratinocytes, on melanocytes is unclear. Other pathogenic genes of DDD include POFUT1, POGLUT1 and PSENEN genes, which is involved in posttranslational modification of Notch receptor. In this study, we aim to determine the ablation of keratinocyte KRT5 affect melanogenesis in melanocyte through Notch signalling pathway. Here we found that KRT5 downregulation decreased the expression of the Notch ligand in keratinocytes and Notch1 intracellular domain in melanocytes, by establishing two cell models of ablation of KRT5 in keratinocytes based on CRISPR/Cas9 site-directed mutation and lentivirus-mediated shRNA. Treatment of melanocytes with Notch inhibitors had same effects with ablation of KRT5 on increase of TYR and decrease of Fascin1. Activation of Notch signalling reverses the effect of ablation of KRT5 on melanogenesis. Immunohistochemistry of DDD lesions with KRT5 gene mutation confirmed changes in the expression of relevant molecules in Notch signalling. Our research elucidates molecular mechanism of KRT5-Notch signalling pathway in the regulation of melanocytes by keratinocytes, and preliminary reveal the mechanism of DDD pigment abnormality caused by KRT5 mutation. These findings identify potential therapeutic targets of the Notch signalling pathway for the treatment of skin pigment disorders.

Notch-Jagged1 signaling and response to bevacizumab therapy in advanced colorectal cancer: A glance to radiomics or back to physiopathology?

The Notch intracellular domain (NICD) and its ligands Jagged-1(Jag1), Delta-like ligand (DLL-3) and DLL4 play an important role in neoangiogenesis. Previous studies suggest a correlation between the tissue levels of NICD and response to therapy with bevacizumab in colorectal cancer (CRC). Another marker that may predict outcome in CRC is radiomics of liver metastases. The aim of this study was to investigate the expression of NICD and its ligands and the role of radiomics in the selection of treatment-naive metastatic CRC patients receiving bevacizumab. Immunohistochemistry (IHC) for NICD, Jag1 and E-cadherin was performed on the tissue microarrays (TMAs) of 111 patients with metastatic CRC treated with bevacizumab and chemotherapy. Both the intensity and the percentage of stained cells were evaluated. The absolute number of CD4+ and CD8+ lymphocytes was counted in three different high-power fields and the mean values obtained were used to determine the CD4/CD8 ratio. The positivity of tumor cells to DLL3 and DLL4 was studied. The microvascular density (MVD) was assessed in fifteen cases by counting the microvessels at 20x magnification and expressed as MVD score. Abdominal CT scans were retrieved and imported into a dedicated workstation for radiomic analysis. Manually drawn regions of interest (ROI) allowed the extraction of radiomic features (RFs) from the tumor. A positive association was found between NICD and Jag1 expression (p< 0.001). Median PFS was significantly shorter in patients whose tumors expressed high NICD and Jag1 (6.43 months vs 11.53 months for negative cases; p = 0.001). Those with an MVD score ≥5 (CD31-high, NICD/Jag1 positive) experienced significantly poorer survival. The radiomic model developed to predict short and long-term survival and PFS yielded a ROC-AUC of 0.709; when integrated with clinical and histopathological data, the integrated model improved the predictive score (ROC-AUC of 0.823). These results show that high NICD and Jag1 expression are associated with progressive disease and early disease progression to anti VEGF-based therapy; the preliminary radiomic analyses show that the integration of quantitative information with clinical and histological data display the highest performance in predicting the outcome of CRC patients.

LY294002 ameliorates psoriatic skin inflammation in mice via blocking the Notch1/Hes1-PTEN/AKT/IL-17A feedback loop.

(IL)-17A, the effective factor of Th17 cells, acts an important pathological role in the pathogenesis of psoriasis. Notch1/hairy and split 1 (Hes1) and PI3K/AKT signaling pathways are interpenetrated and involved in Th17 cell differentiation and IL-17A production. In this present study, we used imiquimod (IMQ)-induced mouse psoriatic skin inflammation to explore the possible mechanism of Notch1/Hes1-PTEN/AKT/IL-17A feedback loop in psoriasis by employing AKT inhibitor LY294002 as an intervention with the methods of flow cytometry analysis, reverse transcription-quantitative polymerase chain reaction, western blot, co-immunoprecipitation, and immunofluorescence. First, LY294002 inhibition can obviously alleviate the mouse psoriatic skin inflammation both in skin structural and histopathological characteristics, which is similar to the changes found in IL-17A antibody-treated mice. Additionally, the interaction between Notch1 intracellular domain (NICD1) and nuclear factor kappa B (NF-κB) activator 1 (Act1) was demonstrated. LY294002 interruption resulted in consistent changes in expression levels of key signaling molecules both in Notch1/Hes1 and PI3K/AKT signaling pathways in a time-dependent manner. Moreover, chloroquine (CQ) can partly reverse the inhibitory effects of LY294002 on the Notch1/Hes1-PTEN/AKT/IL-17A feedback loop by affecting Notch1 ubiquitination and lysosomal degradation. The present study showed that LY294002 can exert the inhibitory effect on Notch1/Hes1-PTEN/AKT/IL-17A feedback loop to regulate Th17 cell differentiation and IL-17A function in the process of psoriasis, which provides a new possible therapeutic strategy for psoriasis.

A small molecule inhibitor of Notch1 modulates stemness and suppresses breast cancer cell growth.

Although breast cancer stem cells (BCSCs) are well characterized, molecularly targeting and eradicating this sub-population remains a challenge in the clinic. Recent studies have explored several signaling pathways that govern stem cell activation: We and others established that the Notch1 signaling plays a significant role in the proliferation, survival, and differentiation of BCSCs. Earlier, we reported that a newly developed small molecule, ASR490, binds to the negative regulatory region (NRR: The activation switch of the Notch receptor) of Notch1. In vitro results demonstrated that ASR490 significantly inhibited BCSCs (ALDH+ and CD44+/CD24-) and breast cancer (BC) growth at nM concentrations, and subsequently inhibited the colony- and mammosphere-forming abilities of BCSCs and BCs. ASR490 downregulated the expressions of Notch1 intracellular domain (NICD: The active form of Notch1) and its downstream effectors Hey1 and HES1. Inhibition of Notch1-NICD facilitated autophagy-mediated growth inhibition by triggering the fusion of autophagosome and autolysosome in BCSCs. ASR490 was found to be non-toxic to healthy cells as compared to existing Notch1 inhibitors. Moreover, oral administration of ASR490 abrogated BCSC and BC tumor growth in the in vivo xenograft models. Together our results indicate that ASR490 is a potential therapeutic agent that inhibits BC tumor growth by targeting and abolishing Notch1 signaling in BCSCs and BC cells.