Downregulation Of Notch Signaling Research Articles

TMIC-61. DEVELOPMENT OF A NOVEL NOTCH-TARGETING ONCOLYTIC HSV FOR GBM TUMORS

Abstract Glioblastoma (GBM) is the most aggressive brain malignancy, which despite continuing worldwide efforts to develop new therapies, remains a deadly disease with limited treatment options. The NOTCH signaling pathway is often hyperactive in GBM tumors, and its activity contributes to tumor progression and resistance to treatment. The role of NOTCH signaling in tumor growth and resistance is well studied, but the significance of this pathway for immunotherapy of GBM is not very well understood. We have recently shown that NOTCH activation following virotherapy contributes to tumor regrowth and induces recruitment of monocytic MDSC cells into the tumor, which limit optimal immune activation to kill tumor cells. For this study, we have discovered a NOTCH interfering intracellular ligand (NIIL) that blocks NOTCH activation from inside glioma cells. RNA sequencing shows the expression of NIIL downregulates NOTCH signaling in vitro. To evaluate the effect of NIIL on virotherapy, we generated an oncolytic herpes simplex virus (oHSV) that also encodes for NIIL. This virus showed a slower spread in vitro but improved therapeutic efficacy in vivo in multiple models of intracranial tumors. In immune competent mice there was a significant improvement in the survival of mice treated with the NIIL-encoding virus, with a fraction of mice showing a complete response after a single treatment. Single-cell RNA sequencing analysis of immune infiltrates is ongoing to understand how NIIL impacts tumor microenvironment and anti-tumor immune therapy.

Deficiency of DDX3X results in neurogenesis defects and abnormal behaviors via dysfunction of the Notch signaling

The molecular mechanisms underlying the neurodevelopmental disorders (NDDs) caused by DDX3X variants remain poorly understood. In this study, we validated that de novo DDX3X variants are enriched in female developmental delay (DD) patients and mainly affect the evolutionarily conserved amino acids based on a meta-analysis of 46,612 NDD trios. We generated a ddx3x deficient zebrafish allele, which exhibited reduced survival rate, DD, microcephaly, adaptation defects, anxiolytic behaviors, social interaction deficits, and impaired spatial recognitive memory. As revealed by single-nucleus RNA sequencing and biological validations, ddx3x deficiency leads to reduced neural stem cell pool, decreased total neuron number, and imbalanced differentiation of excitatory and inhibitory neurons, which are responsible for the behavioral defects. Indeed, the supplementation of L-glutamate or glutamate receptor agonist ly404039 could partly rescue the adaptation and social deficits. Mechanistically, we reveal that the ddx3x deficiency attenuates the stability of the crebbp mRNA, which in turn causes downregulation of Notch signaling and defects in neurogenesis. Our study sheds light on the molecular pathology underlying the abnormal neurodevelopment and behavior of NDD patients with DDX3X mutations, as well as providing potential therapeutic targets for the precision treatment.

Synergistic Effects of Ethanol Extract of Allium sativum (Garlic) with DAPT and ATRA on Notch Signaling Targeted Molecular Action on Lung Cancer Cell line (A549)

Background: The activated oncogenic Notch signalling is an emerging target to treat cancer progression and recurrence. Synthetic inhibitors of Notch receptors are in pre-clinical studies. However, the overexpression of Notch signalling molecules at the gene level needs to be regulated to control cancer progression. Objective: We propose that this can be achieved by gene-regulatory drugs in combination with natural phytochemical compounds. Methods: The ethanol extract of Allium sativum alone and in combination with DAPT and ATRA were evaluated for cytotoxicity on A549 cells by MTT and Trypan blue assays. Their effects on Notch 1, Hes 1 and p53 gene expressions were studied by RT-PCR and qPCR. Their inhibition on metastatic invasion of A549 cells was analyzed by in vitro wound scratch assay. The phytochemicals of the extract were identified by GC-MS analysis. Results: Many organosulfur compounds having anti-cancer potency were identified in GC-MS. The combination treatment with 50μg (IC50) of garlic extract exhibited a highly significant (P≤0.01) synergistic inhibitory effect on A549 cell growth and migration. It has also significantly reduced the expression of Notch 1 and Hes 1 oncogenes and enhanced p53 gene expression, compared with the individual treatments. This indicates the synergistic action of the extract on the downregulation of Notch signalling at the mRNA level. Conclusion: Our study results imply that the combination therapies have potent molecular treatment action via down-regulating Notch signaling target genes and upregulating p53 gene expression as an underlying mechanism of inhibitory action on A549 lung cancer cells.

Corpora cavernosa fibroblasts mediate penile erection.

Penile erection is mediated by the corpora cavernosa, a trabecular-like vascular bed that enlarges upon vasodilation, but its regulation is not completely understood. Here, we show that perivascular fibroblasts in the corpora cavernosa support vasodilation by reducing norepinephrine availability. The effect on penile blood flow depends on the number of fibroblasts, which is regulated by erectile activity. Erection dynamically alters the positional arrangement of fibroblasts, temporarily down-regulating Notch signaling. Inhibition of Notch increases fibroblast numbers and consequently raises penile blood flow. Continuous Notch activation lowers fibroblast numbers and reduces penile blood perfusion. Recurrent erections stimulate fibroblast proliferation and limit vasoconstriction, whereas aging reduces the number of fibroblasts and lowers penile blood flow. Our findings reveal adaptive, erectile activity-dependent modulation of penile blood flow by fibroblasts.

The phosphodiesterase 2A controls lymphatic junctional maturation via cGMP-dependent notch signaling

The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.

Krüppel-like factor 10 modulates stem cell phenotypes of pancreatic adenocarcinoma by transcriptionally regulating notch receptors

BackgroundPancreatic adenocarcinoma (PDAC) is well known for its rapid distant metastasis and local destructive behavior. Loss of Krüppel-like factor 10 (KLF10) contributes to distant migration of PDAC. The role of KLF10 in modulating tumorigenesis and stem cell phenotypes of PDAC is unclear.MethodsAdditional depletion of KLF10 in KC (LSL: KrasG12D; Pdx1-Cre) mice, a spontaneous murine PDAC model, was established to evaluate tumorigenesis. Tumor specimens of PDAC patients were immune-stained of KLF10 to correlate with local recurrence after curative resection. Conditional overexpressing KLF10 in MiaPaCa and stably depleting KLF10 in Panc-1 (Panc-1-pLKO-shKLF10) cells were established for evaluating sphere formation, stem cell markers expression and tumor growth. The signal pathways modulated by KLF10 for PDAC stem cell phenotypes were disclosed by microarray analysis and validated by western blot, qRT-PCR, luciferase reporter assay. Candidate targets to reverse PDAC tumor growth were demonstrated in murine model.ResultsKLF10, deficient in two-thirds of 105 patients with resected pancreatic PDAC, was associated with rapid local recurrence and large tumor size. Additional KLF10 depletion in KC mice accelerated progression from pancreatic intraepithelial neoplasia to PDAC. Increased sphere formation, expression of stem cell markers, and tumor growth were observed in Panc-1-pLKO-shKLF10 compared with vector control. Genetically or pharmacologically overexpression of KLF10 reversed the stem cell phenotypes induced by KLF10 depletion. Ingenuity pathway analysis and gene set enrichment analysis showed that Notch signaling molecules, including Notch receptors 3 and 4, were over-expressed in Panc-1-pLKO-shKLF10. KLF10 transcriptionally suppressed Notch-3 and -4 by competing with E74-like ETS transcription factor 3, a positive regulator, for promoter binding. Downregulation of Notch signaling, either genetically or pharmacologically, ameliorated the stem cell phenotypes of Panc-1-pLKO-shKLF10. The combination of metformin, which upregulated KLF10 expression via phosphorylating AMPK, and evodiamine, a non-toxic Notch-3 methylation stimulator, delayed tumor growth of PDAC with KLF10 deficiency in mice without prominent toxicity.ConclusionsThese results demonstrated a novel signaling pathway by which KLF10 modulates stem cell phenotypes in PDAC through transcriptionally regulating Notch signaling pathway. The elevation of KLF10 and suppression of Notch signaling may jointly reduce PDAC tumorigenesis and malignant progression.

Abstract 5828: Distinct immunosuppressive tumor microenvironment modulates small cell lung carcinoma neuroendocrine phenotypic plasticity and heterogeneity

Abstract Small cell lung carcinoma (SCLC) is prototypical of cancers with exceptional chemoresistance, and metastatic capabilities fueled by remarkable heterogeneity. The cell-extrinsic factors that govern SCLC heterogeneity are poorly understood especially since non-genetic mechanisms are thought to be major contributors to its intratumoral heterogeneity and plasticity. In order to answer the above question, we sought to inquire about the role of tumor microenvironment (TME) in SCLC heterogeneity which is challenged universally by a lack of patient-derived adequate tumor biopsies. Leveraging large tumor sampling performed at rapid autopsies done on 10 different patients who died from SCLC metastatic disease, we developed a multi-omics approach involving spatial transcriptomics (ST) (Whole transcriptomic atlas), bulk whole genome sequencing, and multiplex immunofluorescence (IF) and standard immunohistochemistry (IHC) to better characterize spatially proximate tumor (36 segments), TME (32 segments) and 4 normal segments. Genomically, SCLC characteristic alterations like TP53 (70%) and RB1 (80%) inactivation and MYC (50%)/MYCL (80%) were evident. ST profiling reveals three distinct tumor subtypes corresponding to the known high-Neuroendocrine (NE) and low-NE SCLC subtypes as well as a novel hybrid-NE state with multiple elevated cancer hallmark capabilities and morphological evidence of nesting and budding. High-NE and hybrid-NE subtypes appear to be driven by ASCL1 and the low-NE subtype resembles the SCLC- inflamed subtype. ST profiling of proximate TMEs reveals similar heterogeneity that is closely linked with SCLC-NE heterogeneity at inter-patient and intra-tumoral levels with distinct features and cellular compositions. Specifically, low-NE TME is marked by increased pro and anti-tumor features including B cells, NK cells, M1-macrophages, and evidence of more checkpoint molecules. High-NE TMEs are predominated by a distinct absence of adaptive immune cells and a strong presence of M2 macrophages. Most strikingly the hybrid-NE TME shows a significant enrichment of immunosuppressive cancer-associated fibroblasts with the absence of T and other adaptive immune cells confirmed using IHC and IF. Next, we attempt to narrow down the fibroblast growth factor receptor (FGF) axis as one of the unique TME-driven factor(s) that could groom non-NE features in SCLC fueling their plasticity and heterogeneity. Subsequently using various SCLC cell lines, we demonstrate that in-vitro modulation of FGFR in SCLC may constrain their plasticity by downregulation of NOTCH signaling, MYC, and its downstream effector REST. We describe the remarkable phenotypic heterogeneity of SCLC tumor and TME and demonstrate their interlinking as well as potential translational avenues of leveraging the cellular crosstalk in order to constrain SCLC heterogeneity. Citation Format: Parth Anil Desai, Nobuyuki Takahashi, Yingying Cao, Christopher Schultz, Darryl Nousome, Desiree Tillo, Lakshya Chauhan, Mayank tandon, Rajesh kumar Bhardwaj, Delphine Lissa, Vinodh Rajapakse, Suresh kumar, Yang Zhang, Elmeskini Rajaa, Donna Butcher, Linda Scuito, Jingjing Gong, Brett Schroder, Andrew Warner, Samantha Nichols, Jordan Kimberly, Alejandro Schaffer, Mohit kumar Jolly, Stephen Hewitt, Anish Thomas. Distinct immunosuppressive tumor microenvironment modulates small cell lung carcinoma neuroendocrine phenotypic plasticity and heterogeneity [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 5828.

Roxadustat induces hepatotoxicity in zebrafish embryos via inhibiting Notch signaling.

Roxadustat is a novel and effective small-molecule inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PHI). However, little research has been done on its toxicity to vertebrate embryonic development. In this study, we used zebrafish to assess the effects of roxadustat on early embryonic development. Exposure to 14, 28, and 56 μM roxadustat resulted in abnormal embryonic development in zebrafish embryos, such as shortened body length and early liver developmental deficiency. Roxadustat exposure resulted in liver metabolic imbalance and abnormal liver tissue structure in adult zebrafish. In addition, roxadustat could up-regulate oxidative stress, and astaxanthin (AS) could partially rescue liver developmental defects by down-regulation of oxidative stress. After exposure to roxadustat, the Notch signaling is down-regulated, and the use of an activator of Notch signaling can partially rescue hepatotoxicity. Therefore, our research indicates that roxadustat may induce zebrafish hepatotoxicity by down-regulating Notch signaling. This study provides a reference for the clinical use of roxadustat.

Pinostrobin suppresses the proliferation of lung carcinoma cells by abrogating the cell cycle progression through the inhibition of Notch signaling pathway

• Pinostrobin (PN) suppressed the growth and proliferation of lung cancer cells (A549). • PN induced apoptosis via intrinsic apoptosis pathway. • PN abrogated the cell cycle progression in A549 lung cancer cells. • PN downregulated notch signaling cascade in lung cancer. Pinostrobin (PN) is a dietary bioflavonoid naturally found in numerous medicinal plants. Even though the anticancer potential of flavonoids has been studied; however the molecular targets and the exact mechanism behind their apoptosis-inducing actions are not yet fully elucidated. The present study was aimed to investigate if PN could induce apoptosis in lung cancer A549 cells of human origin. PN reduced the proliferation of A549 cells in a dose-dependent manner. Induction of apoptosis was observed in PN-treated lung cancer cells as evident by DNA fragmentation and Annexin V positive cells. The PN induced reactive oxygen species (ROS) generation in A549 cells, leading to activation of key regulators of apoptosis such as Bax, Bad and Bcl2 as well as release of cytochrome c, and activation of the caspase cascade including caspase-9 and -3. Enhanced loss of Δψm, cytochrome c release and cleavage of caspase-3 & -9 strongly corroborated our findings. Furthermore, PN treatment of lung cancer cells arrested the cell cycle in G0/G1 phase, inhibited constitutive expression of Cyclin D1and CDK4 and induced the mRNA levels of p21 Cip1 . A dose-dependent decreased expression of Notch-1, Jagged-1 and Hes-1 were noted in PN treated A549 cells. These findings demonstrated that PN suppressed the growth of A549 cells via downregulating the Notch pathway. Thus, subsequent studies focusing on molecular mechanism of PN can pave path for anticancer drug design.

Carbon Ion Irradiation Downregulates Notch Signaling in Glioma Cell Lines, Impacting Cell Migration and Spheroid Formation.

Photon-based radiotherapy upregulates Notch signaling in cancer, leading to the acquisition of the stem cell phenotype and induction of invasion/migration, which contributes to the development of resistance to therapy. However, the effect of carbon ion radiotherapy (CIRT) on Notch signaling in glioma and its impact on stemness and migration is not explored yet. Human glioma cell lines (LN229 and U251), stable Notch1 intracellular domain (N1ICD) overexpressing phenotype of LN229 cells, and Notch inhibitor resistant LN229 cells (LN229R) were irradiated with either photon (X-rays) or (carbon ion irradiation) CII, and expressions of Notch signaling components were accessed by RT-PCR, Western blotting, and enzymatic assays and flow cytometry. Spheroid forming ability, cell migration, and clonogenic assay were used to evaluate the effect of modulated Notch signaling by irradiation. Our results show that X-ray irradiation induced the expression of Notch signaling components such as Notch receptors, target genes, and ADAM17 activity, while CII reduced it in glioma cell lines. The differential modulation of ADAM17 activity by CII and X-rays affected the cell surface levels of NOTCH1 and NOTCH2 receptors, as they were reduced by X-ray irradiation but increased in response to CII. Functionally, CII reduced the spheroid formation and migration of glioma cells, possibly by downregulating the N1ICD, as stable overexpression of N1ICD rescued these inhibitory effects of CII. Moreover, LN229R that are less reliant on Notch signaling for their survival showed less response to CII. Therefore, downregulation of Notch signaling resulting in the suppression of stemness and impaired cell migration by CII seen here may reduce tumor regrowth and disease dissemination, in addition to the well-established cytotoxic effects.

Biophysical Regulations of Epigenetic State and Notch Signaling in Neural Development Using Microgroove Substrates.

A number of studies have recently shown how surface topography can alter the behavior and differentiation patterns of different types of stem cells. Although the exact mechanisms and molecular pathways involved remain unclear, a consistent portion of the literature points to epigenetic changes induced by nuclear remodeling. In this study, we investigate the behavior of clinically relevant neural populations derived from human pluripotent stem cells when cultured on polydimethylsiloxane microgrooves (3 and 10 μm depth grooves) to investigate what mechanisms are responsible for their differentiation capacity and functional behavior. Our results show that microgrooves enhance cell alignment, modify nuclear geometry, and significantly increase cellular stiffness, which we were able to measure at high resolution with a combination of light and electron microscopy, scanning ion conductance microscopy (SICM), and atomic force microscopy (AFM) coupled with quantitative image analysis. The microgrooves promoted significant changes in the epigenetic landscape, as revealed by the expression of key histone modification markers. The main behavioral change of neural stem cells on microgrooves was an increase of neuronal differentiation under basal conditions on the microgrooves. Through measurements of cleaved Notch1 levels, we found that microgrooves downregulate Notch signaling. We in fact propose that microgroove topography affects the differentiation potential of neural stem cells by indirectly altering Notch signaling through geometric segregation and that this mechanism in parallel with topography-dependent epigenetic modulations acts in concert to enhance stem cell neuronal differentiation.

Leptin modulated microRNA-628-5p targets Jagged-1 and inhibits prostate cancer hallmarks

MicroRNAs (miRNAs) are single-stranded non-coding RNA molecules that play a regulatory role in gene expression and cancer cell signaling. We previously identified miR-628-5p (miR-628) as a potential biomarker in serum samples from men with prostate cancer (PCa) (Srivastava et al. in Tumour Biol 35:4867–4873, 10.1007/s13277-014-1638-1, 2014). This study examined the detailed cellular phenotypes and pathways regulated by miR-628 in PCa cells. Since obesity is a significant risk factor for PCa, and there is a correlation between levels of the obesity-associated hormone leptin and PCa development, here we investigated the functional relationship between leptin and miR-628 regulation in PCa. We demonstrated that exposure to leptin downregulated the expression of miR-628 and increased cell proliferation/migration in PCa cells. We next studied the effects on cancer-related phenotypes in PCa cells after altering miR-628 expression levels. Enforced expression of miR-628 in PCa cells inhibited cell proliferation, reduced PCa cell survival/migration/invasion/spheroid formation, and decreased markers of cell stemness. Mechanistically, miR-628 binds with the JAG1-3′UTR and inhibits the expression of Jagged-1 (JAG1). JAG1 inhibition by miR-628 downregulated Notch signaling, decreased the expression of Snail/Slug, and modulated epithelial-mesenchymal transition and invasiveness in PC3 cells. Furthermore, expression of miR-628 in PCa cells increased sensitivity towards the drugs enzalutamide and docetaxel by induction of cell apoptosis. Collectively our data suggest that miR-628 is a key regulator of PCa carcinogenesis and is modulated by leptin, offering a novel therapeutic opportunity to inhibit the growth of advanced PCa.

Abstract 5822: Leptin modulated microRNA-628-5p targets Jagged1 and inhibits prostate cancer hallmarks

Abstract Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer and the second leading cause of cancer deaths in men. Obesity is a major risk factor for prostate cancer. Leptin is an obesity hormone that controls body weight by regulating energy intake and expenditure, and clinical studies have demonstrated a correlation between blood leptin levels and PCa development. MicroRNAs (miRs) are master regulators of cancer cell signaling, including in advanced PCa. We previously identified miR-628-5p as a potential biomarker in PCa patients that was downregulated in serum samples of men with PCa (Tumor Biol., 2014, 35: 4867). In this study, we investigated the link between the obesity hormone leptin and the functional role of miR-628-5p in PCa cell lines. We demonstrate that leptin downregulates the expression of miR-628-5p and increases cell proliferation and migration in PCa cells. Enforced expression of miR-628-5p inhibited cell proliferation in PCa cells. Further, miR-628-5p reduced PCa cell survival/migration/invasion/spheroids formation and cell stemness. Mechanistically, miR-628-5p binds with JAG1-3’UTR and inhibits the expression of Jagged-1 (JAG1). JAG1 inhibition by miR-628-5p also downregulated Notch signaling, decreased the expression of snail/slug, and modulated epithelial-mesenchymal transition and invasiveness in PC3 cells. Furthermore, miR-628-5p increased sensitivity towards enzalutamide and docetaxel by inducing cell apoptosis. Collectively our data suggested that miR-628-5p is a key regulator of PCa carcinogenesis modulated by adipokine leptin and offers a novel therapeutic opportunity to inhibit the growth of advanced PCa. Citation Format: Leslimar Rios Colon, Juliet Chijioke, Suryakant Niture, Zainab Afzal, Qi Qi, Anvesha Srivastava, Malathi Ramalinga, Habib Kedir, Patrice Cagle, Elena Arthur, Gagan Deep, Simeng Suy, Sean Collins, Deepak Kumar. Leptin modulated microRNA-628-5p targets Jagged1 and inhibits prostate cancer hallmarks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5822.

Oroxylin A attenuates cytokines in neonatal asthmatic mice by regulating Notch signaling pathway


 
 
 
 Purpose: To investigate the protective effect of oroxylin A against neonatal asthma in mice.
 Methods: Asthma was induced in P12 mice with ovalbumin (OVA) immediately after litter (day 0), and on day 14 of protocol. Oroxylin A was given daily in two doses (2 and 10 mg/kg, i.p.) from day 0 to 14, 2 h before the administration of OVA. The protective effect of oroxylin A against asthma was determined by evaluating lung function and levels of cytokines and IgE in OVA-challenged mice. Moreover, Western blot assay and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the effect of oroxylin A on Notch signaling pathway in OVA-challenged mice.
 Results: Oroxylin A improved lung function in OVA-challenged mice. There were significant (p < 0.01) reductions in levels of IgE and cytokines in the BALF of oroxylin A-treated group, when compared to OVA group of mice. Furthermore, treatment with oroxylin A significantly ameliorated the OVA-induced changes in protein expressions of Notch1, Jagged1, NICD, PTEN, PI3K and Akt in the CD+ cells of OVA-challenged mice (p < 0.01).
 Conclusion: These results indicate that oroxylin A improves lung function and reduces the level of cytokines in neonatal asthmatic rat by downregulating Notch signaling. Thus, oroxylin A may be clinically beneficial in the management of asthma.
 
 
 

Lenvatinib induces cardiac developmental toxicity in zebrafish embryos through regulation of Notch mediated-oxidative stress generation.

Due to an increasing number of abused drugs dumped into the wastewater, more and more drugs are detected in the water environment, which may affect the survival of aquatic organisms. Lenvatinib is a multi-targeted tyrosine kinase inhibitor, and is clinically used to treat differentiated thyroid cancer, renal epithelial cell carcinoma and liver cancer. However, there are few reports on the effects of lenvatinib in embryos development. In this study, zebrafish embryos were used to evaluate the effect of lenvatinib on cardiovascular development. Well-developed zebrafish embryos were selected at 6h post fertilization (hpf) and exposed to 0.05 mg/L, 0.1mg/L and 0.2mg/L lenvatinib up to 72 hpf. The processed embryos demonstrated cardiac edema, decreased heart rate, prolonged SV-BA distance, inhibited angiogenesis, and blocked blood circulation. Lenvatinib caused cardiac defects in the whole stage of cardiac development and increased the apoptosis of cardiomyocyte. Oxidative stress in the processed embryos was accumulated and inhibiting oxidative stress could rescue cardiac defects induced by lenvatinib. Additionally, we found that lenvatinib downregulated Notch signaling, and the activation of Notch signaling could rescue cardiac developmental defects and downregulate oxidative stress level induced by lenvatinib. Our results suggested that lenvatinib might induce cardiac developmental toxicity through inducing Notch mediated-oxidative stress generation, raising concerns about the harm of exposure to lenvatinib in aquatic organisms.

Danzhi Xiaoyao Powder Promotes Neuronal Regeneration by Downregulating Notch Signaling Pathway in the Treatment of Generalized Anxiety Disorder

Background: Generalized anxiety disorder (GAD) is one of the most common types of anxiety disorders with unclear pathogenesis. Our team’s previous research found that extensive neuronal apoptosis and neuronal regeneration disorders occur in the hippocampus of GAD rats. Danzhi Xiaoyao (DZXYS) Powder can improve the anxiety behavior of rats, but its molecular mechanism is not well understood. Objective: This paper discusses whether the pathogenesis of GAD is related to the abnormal expression of Notch signal pathway, and whether the anti-anxiety effect of DZXYS promotes nerve regeneration in the hippocampus by regulating the Notch signaling pathway. Methods: The animal model of GAD was developed by the chronic restraint stress and uncertain empty bottle stimulation method. After the model was successfully established, the rats in the model preparation group were divided into the buspirone, DZXYS, DZXYS + DAPT, and model groups, and were administered the corresponding drug intervention. The changes in body weight and food intake of rats were continuously monitored throughout the process. The changes in anxiety behavior of rats were measured by open field experiment and elevated plus-maze test, and morphological changes and regeneration of neurons in the rat hippocampus were observed by HE staining and double immunofluorescence staining. Changes in the expression of key targets of the Notch signaling pathway in the hippocampus were monitored by real-time fluorescence quantitative PCR and western blotting. Results: In this study, we verified that the GAD model was stable and reliable, and found that the key targets of the Notch signaling pathway (Notch1, Hes1, Hes5, etc.) in the hippocampus of GAD rats were significantly upregulated, leading to the increased proliferation of neural stem cells in the hippocampus and increased differentiation into astrocytes, resulting in neuronal regeneration. DZXYS intervention in GAD rats can improve appetite, promote weight growth, and significantly reverse the anxiety behavior of GAD rats, which can inhibit the upregulation of key targets of the Notch signaling pathway, promote the differentiation of neural stem cells in the hippocampus into neurons, and inhibit their differentiation into astrocytes, thus alleviating anxiety behavior. Conclusion: The occurrence of GAD is closely related to the upregulation of the Notch signaling pathway, which hinders the regeneration of normal neurons in the hippocampus, while DZXYS can downregulate the Notch signaling pathway and promote neuronal regeneration in the hippocampus, thereby relieving anxiety behavior.

Silencing of Fused Toes Homolog (FTS) Increases Radiosensitivity to Carbon-Ion Through Downregulation of Notch Signaling in Cervical Cancer Cells.

The effects of Carbon ion radiation (C-ion) alone or in combination with fused toes homolog (FTS) silencing on Notch signaling were investigated in uterine cervical cancer cell lines (ME180 and CaSki). In both cell lines, upon irradiation with C-ion, the expression of Notch signaling molecules (Notch1, 2, 3 and cleaved Notch1), γ-secretase complex molecules and FTS was upregulated dose-dependently (1, 2 and 4 Gy) except Notch1 in ME180 cells where the change in expression was not significant. However, overexpression of these molecules was attenuated upon silencing of FTS. The spheroid formation, expression of stem cell markers (OCT4A, Sox2 and Nanog) and clonogenic cell survival were reduced by the combination as compared to FTS silencing or C-ion irradiation alone. Additionally, immunoprecipitation and immunofluorescence assay revealed interaction and co-localization of FTS with Notch signaling molecules. In conclusion, FTS silencing enhances the radio-sensitivity of the cervical cancer cells to C-ion by downregulating Notch signaling molecules and decreasing the survival of cancer stem cells.

Canonical Notch Signaling Is Required for KrasG12D -Induced Myeloproliferative Neoplasm

Notch is a highly conserved signaling pathway that regulates cell-fate specification and tissue homeostasis in various contexts. Depending on the context, Notch signaling could be oncogenic or tumor suppressive. The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function (GOF) NOTCH1 mutations are prevalent in human T cell leukemia, while loss of Notch signaling is reported in myeloid leukemias.To investigate the role of Notch signaling in T cell acute lymphoblastic leukemia/lymphoma (T-ALL) and myeloproliferative neoplasm (MPN) induced by Ras mutation, we downregulated Notch signaling in oncogenic Kras (KrasG12D) mouse model, which develops T-ALL and MPN simultaneously. We took two independent genetic approaches to downregulate Notch signaling. Mx1-Cre-mediated conditional expression of dominant-negative Mastermind-like1 (DNMAML) potently inhibits canonical Notch-mediated transcriptional activation, while knocking out Protein O-fucosyltransferase 1 (Pofut1) reduces interactions of Notch receptors and their ligands and downregulates Notch signaling. To study the cell-autonomous functions of downregulating Notch signaling in oncogenic Kras -induced leukemogenesis, we transplanted either bone marrow cells or splenocytes into lethally irradiated mice.As expected, inhibition of Notch signaling significantly inhibited T-cell development and completely prevented T-ALL development in recipients; T-ALL that developed in a fraction of recipient mice were derived from rare donor cells that expressed oncogenic Kras, preserved intact Notch signaling, and acquired GOF Notch1 mutation(s). Surprisingly, we found that downregulation of Notch signaling inhibited MPN development in a cell-autonomous manner. Analysis of donor-derived hematopoiesis revealed that loss of Notch signaling significantly reduced myeloid compartment in Kras recipients with significant decrease of proliferation. Further mechanistic studies indicate that inhibition of Notch signaling significantly upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. RNA-Seq analysis of control and mutant myeloid progenitors (MPs) demonstrated that KrasG12D MPs exhibited enhanced oxidative phosphorylation and mitochondria respiration, and this aberrant gene expression pattern was largely restored by DNMAML expression. These metabolic changes were further validated in functional assays. Furthermore, we demonstrated that combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target Notch downstream mechanisms in oncogenic Ras-driven myeloid leukemias. DisclosuresPadron:Incyte: Honoraria, Research Funding.

ADAM10 is involved in the oncogenic process and chemo-resistance of triple-negative breast cancer via regulating Notch1 signaling pathway, CD44 and PrPc

BackgroundTriple-negative breast cancer (TNBC) is the most challenging breast cancer subtype to treat, because it is so aggressive with shorter survival. Chemotherapy remains the standard treatment due to the lack of specific and effective molecular targets. The aim of the present study is to investigate the potential roles of A Disintegrin and Metalloproteinase 10 (ADAM10) on TNBC cells and the effects of combining ADAM10 expression and neoadjuvant chemotherapy treatment (NACT) to improve the overall survival in breast cancer patients.MethodsUsing a series of breast cancer cell lines, we measured the expression of ADAM10 and its substrates by quantitative real-time PCR assay (qRT-PCR) and western blot analysis. Cell migration and invasion, cell proliferation, drug sensitivity assay, cell cycle and apoptosis were conducted in MDA-MB-231 cells cultured with ADAM10 siRNA. The effect of ADAM10 down-regulation by siRNA on its substrates was assessed by western blot analysis. We performed immunohistochemical staining for ADAM10 in clinical breast cancer tissues in 94 patients receiving NACT.ResultsThe active form of ADAM10 was highly expressed in TNBC cell lines. Knockdown of ADAM10 in MDA-MB-231 cells led to a significant decrease in cell proliferation, migration, invasion and the IC50 value of paclitaxel and adriamycin, while induced cell cycle arrest and apoptosis. And these changes were correlated with down-regulation of Notch signaling, CD44 and cellular prion protein (PrPc). In clinical breast cancer cases, a high ADAM10 expression in pre-NACT samples was strongly associated with poorer response to NACT and shorter overall survival.ConclusionsThese data suggest the previously unrecognized roles of ADAM10 in contributing to the progression and chemo-resistance of TNBC.

Abstract 129: Apoptosis & anti-proliferative nature of Aescin on 7,12 dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinogenesis via downregulating Notch signaling

Abstract Worldwide, oral squamous cell carcinoma (OSCC) is the sixth most common cancer & ranked as second most common cancer in India. The most implicated etiologic factors in the development of oral cancer is the use of tobacco, alcohol & also HPV infection. Aescin, a pentacyclic triterpenoid saponin mixtures derived from Aesculus hippocastanum L (horse chestnut) that has been used as traditional herbal medicine, exists the pharmacodynamic properties, anti- edematous, anti-inflammatory & antitumor. The present study, Tumors were induced by applying 0.5% DMBA topically to hamster buccal pouch (HBP) thrice a week for 14 weeks & Aescin was orally administered at a various dose of 40 mg/kg body weight on alternate days of DMBA painting. We observed, reduced the tumor incidence, diminished lipid peroxidation, & elevation of antioxidants, liver marker enzymes by Aescin. Furthermore, aescin induced apoptosis in DMBA induced tumor bearing animals significantly inhibited cell to cell communication of Notch-1, Notch-2 & its ligand & Jagged-1 leads to inhibited expression of the Notch downstream signaling target Hes-1 & Hey-1 analyzed by immunohistochemical analysis. These results clearly suggest that Aescin prevents lipid peroxidation, protects the antioxidant defense system, prohibit the dysplasia & anti-carcinogenic potential. we concluded; the results provide an important new insight of Notch signaling pathway & aescin might be developed as a potential chemo preventive agent for oral cancer. Key words: Oral cancer, aescin, antioxidant, apoptosis, chemopreventive, Notch signaling Citation Format: Hemavardhini Ramadoss, Suresh Kathiresan, Sanjay Gupta. Apoptosis & anti-proliferative nature of Aescin on 7,12 dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinogenesis via downregulating Notch signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 129.