Activation Of Notch Signaling Pathway Research Articles

Human umbilical vein endothelial cells derived-exosomes promote osteosarcoma cell stemness by activating Notch signaling pathway

ABSTRACT Osteosarcoma is one of the most common primary malignant tumors of bone in adolescents. Human umbilical vein endothelial cells (HUVECs) derived exosomes are associated with osteosarcoma cell stemness. Little is known about the function of HUVECs-exosomes in osteosarcoma cell stemness. This work aimed to investigate the mechanism of action of HUVECs-exosomes in regulating stem cell-like phenotypes of osteosarcoma cells. HUVECs were treated with GW4869 (exosome inhibitor). Human osteosarcoma cells (U2OS and 143B) were treated with HUVECs supernatant, HUVECs-exosomes with or without RO4929097 (γ secretase inhibitor, used to block Notch signaling pathway). We found that HUVECs supernatant and HUVECs-exosomes enhanced the proportions of STRO-1+CD117+ cells and the expression of stem cell-related proteins Oct4 and Sox2. Both HUVECs supernatant and HUVECs-exosomes promoted the sarcosphere formation efficiency of U2OS and 143B cells. These stem-like phenotypes of U2OS and 143B cells conferred by HUVECs-exosomes were repressed by GW4869. Moreover, HUVECs-exosomes promoted the expression of Notch1, Hes1 and Hey1 in the U2OS and 143B cells. RO4929097 treatment reversed the impact of HUVECs-exosomes on Notch1, Hes1, and Hey1 expression by inhibiting Notch1 signaling pathway. In conclusion, this work demonstrated that HUVECs-exosomes promoted cell stemness in osteosarcoma through activating Notch signaling pathway. Thus, our data reveal the mechanism of HUVECs-exosomes in regulating cell stemness of osteosarcoma, and provide a theoretical basis for osteosarcoma treatment by exosomes.

Cardiac developmental toxicity and transcriptome analyses of zebrafish (Danio rerio) embryos exposed to Mancozeb

Mancozeb (MZ), an antibacterial pesticide, has been linked to reproductive toxicity, neurotoxicity, and endocrine disruption. However, whether MZ has cardiactoxicity is unclear. In this study, the cardiotoxic effects of exposure to environment-related MZ concentrations ranging from 1.88 μM to 7.52 μM were evaluated at the larval stage of zebrafish. Transcriptome sequencing predicted the mechanism of MZ-induced cardiac developmental toxicity in zebrafish by enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Consistent with morphological changes, the osm, pfkfb3, foxh1, stc1, and nrarpb genes may effect normal development of zebrafish heart by activating NOTCH signaling pathways, resulting in pericardial edema, myocardial fibrosis, and congestion in the heart area. Moreover, differential gene expression analysis indicated that cyp-related genes (cyp1c2 and cyp3c3) were significantly upregulated after MZ treatment, which may be related to apoptosis of myocardial cells. These results were verified by real-time quantitative RT-qPCR and acridine orange staining. Our findings suggest that MZ-mediated cardiotoxic development of zebrafish larvae may be related to the activation of Notch and apoptosis-related signaling pathways.

Spindle and kinetochore-associated complex subunit 3 (SKA3) promotes stem cell-like properties of hepatocellular carcinoma cells through activating Notch signaling pathway.

BackgroundCancer stemness contributes to hepatocellular carcinoma (HCC) initiation, metastasis, drug resistance, and recurrence. The spindle and kinetochore-associated (SKA) complex has been shown to be involved in tumor progression; however, its effects on cancer stem cell-like properties have not yet been examined. This research sought to study each subunit of the SKA complex in HCC systematically.MethodsBioinformatic analyses were carried out to examine the expression and clinical data of the SKA complex’s each subunit in HCC. The expression of the target genes was detected by quantitative reverse transcription-polymerase chain reaction and Western blot assays. Clone formation and Transwell assays were performed to assess the proliferation and migration abilities of the SKA complex’s each subunit. Sphere formation assays and subcutaneous xenograft experiments were performed to investigate the effects of SKA complex subunit 3 (SKA3) on the self-renewal and tumorigenic abilities of HCC.ResultsEach subunit of the SKA complex was highly expressed in HCC, but only SKA complex subunit 1 (SKA1) and SKA3 were associated with the poor overall survival of HCC patients. Additionally, the HCC cells overexpressing SKA3 exhibited increased migration, invasion, proliferation, self-renewal, Sorafenib resistance and tumorigenic abilities. Notch signaling played a vital role in the process by which SKA3 promoted HCC stemness.ConclusionsSKA3 promotes HCC stem cell-like properties via the Notch signaling pathway. As SKA3 appears to act as a regulator of stemness in HCC, it might be a potential molecular target for HCC.

Bromodomain-containing protein 9 is a prognostic biomarker associated with immune infiltrates and promotes tumor malignancy through activating notch signaling pathway in negative HIF-2α clear cell renal cell carcinoma.

HIF-2α selective inhibitor showed successful efficacy in sensitive clear cell renal cell carcinoma (ccRCC) presenting higher levels of HIF-2α compared to resistant tumors with low level of HIF-2α (negative HIF-2α ccRCC). Currently, negative HIF-2α ccRCC lacks truly effective therapeutic agents to improve the outcomes. Bromodomain-containing protein 9 (BRD9) plays a critical role in human hepatocellular carcinoma, squamous cell lung cancer, acute myeloid leukemia, and so on. However, expression and biological role of BRD9 in negative HIF-2α ccRCC is poorly understood. Clinically, we demonstrated that expression of BRD9 in negative HIF-2α ccRCC tissues was higher than that in positive HIF-2α ccRCC. Moreover, high BRD9 expression was correlated with unfavorable clinicopathological features and predicted the poor overall survival of negative HIF-2α ccRCC patients. Functionally, BRD9 knockout resulted in reduced proliferation, migration and invasion of negative HIF-2α ccRCC cells (Caki-2). In addition, BRD9 was related to the TIIC infiltration level in negative HIF-2α ccRCC tissues. Mechanistically, Gene set enrichment analysis (GSEA) showed that BRD9 was closely related to Notch signaling pathway. BRD9 knockout resulted in reduced mRNA level of Hes1 and Notch1 in negative HIF-2α ccRCC in vitro. The overexpression of NICD (Notch intracellular domain) enhanced malignant behaviors of Caki-2 cells with BRD9 knockout. And Notch inhibition led to attenuation of cell growth and reduced migration and invasion in Caki-2 cells. Overall, our results identified that BRD9 promotes the proliferation, migration and invasion of negative HIF-2α ccRCC cells by targeting Notch signaling pathway and serve as a promising biomarker for negative HIF-2α ccRCC.

Decreased autophagy impairs osteogenic differentiation of adipose-derived stem cells via Notch signaling in diabetic osteoporosis mice

BackgroundThe osteogenic differentiation ability of adipose-derived stem cells (ASCs) is attenuated in type 2 diabetic osteoporosis (Dop) mice. Several studies suggest autophagy and Notch signaling pathway play vital roles in cell proliferation, differentiation, and osteogenesis. However, the mechanisms of autophagy and Notch signaling in the osteogenic differentiation of Dop ASCs were unclear. Thus, it is meaningful to reveal potential correlations between autophagy, Notch signaling, and osteogenesis, and explore involved molecular mechanisms in Dop ASCs. Materials and methodsThe diabetic osteoporosis C57BL/6 mouse model, which was confirmed by micro-CT and HE & Masson staining, was established through high-sugar and high-fat diet and streptozotocin injection. ASCs were obtained from the inguinal subcutaneous fat of Dop mice. The multi-differentiation potential of ASCs was evaluated by staining with Alizarin Red (osteogenesis), Oil Red O (adipogenesis), and Alcian blue (chondrogenesis). Cell viability was assessed by Cell Counting Kit-8 assay. Torin1, an inhibitor of mTOR, was used to stimulate the autophagy signaling pathway. DAPT, a γ-secretase inhibitor, was used to suppress Notch signaling pathway activity. Gene and protein expression of autophagy, Notch signaling pathway, and osteogenic factors were detected by real-time quantitative PCR, western blot, and immunofluorescence microscopy. ResultsOur findings showed autophagy and osteogenic differentiation ability of Dop ASCs exhibited downward trends that were both rescued by Torin1. Notch signaling was suppressed in Dop ASCs, but upregulated when autophagy was activated. After activation of autophagy, DAPT treatment led to decreased Notch signaling pathway activation and attenuated osteogenic differentiation ability in Dop ASCs. ConclusionsDownregulated autophagy suppressed Notch signaling, leading to a reduced osteogenic differentiation capacity of Dop ASCs, and Torin1 can rescue this process by activating autophagy. Our findings contribute to understanding the mechanism underlying impairment of the osteogenic differentiation ability of Dop ASCs.

EGCG inhibits growth of tumoral lesions on lip and tongue of K-Ras transgenic mice through the Notch pathway

Epigallocatechin-3-gallate (EGCG), the main active ingredient of green tea, exhibits low toxic side effect and versatile bioactivities, and its anti-cancer effect has been extensively studied. Most of the studies used cancer cell lines and xenograft models. However, whether EGCG can prevent tumor onset after cancer-associated mutations occur is still controversial. In the present study, Krt14-cre/ERT-Kras transgenic mice were developed and the expression of K-RasG12D was induced by tamoxifen. Two weeks after induction, the K-Ras mutant mice developed exophytic tumoral lesions on the lips and tongues, with significant activation of Notch signaling pathway. Administration of EGCG effectively delayed the time of appearance, decreased the size and weight of tumoral lesions, relieved heterotypic hyperplasia of tumoral lesions, and prolonged the life of the mice. The Notch signaling pathway was significantly inhibited by EGCG in the tumoral lesions. Furthermore, EGCG significantly induced cell apoptosis and inhibited the proliferation of tongue cancer cells by blocking the activation of Notch signaling pathway. Taken together, these results indicate EGCG as an effective chemotherapeutic agent for tongue cancer by targeting Notch pathway.

LINC01355 Contributes to Malignant Phenotype of Oral Squamous Cell Carcinoma and Cytotoxic T Cell Infiltration via Activating Notch Signaling Pathway.

LINC01355 has been demonstrated to be dysregulated in several cancers. However, the exact molecular function of LINC01355 in the pathogenesis of OSCC remains unstudied. Here, we reported the effect of LINC01355 in OSCC and investigated the mechanisms. Firstly, we found that the results indicated LINC01355 was increased in OSCC cells. Knockdown of LINC01355 repressed OSCC cell proliferation, migration, and invasion. Recently, immunotherapy is a significant method for the treatment of cancers, in which CD8+ T cells exhibit a significant role. The influence of LINC01355 on the antitumor activity of CD8+ T cells was also focused in this study. As shown, the silence of LINC01355 could repress OSCC tumor growth via inducing CD8+ T cell immune responses. In addition, we found that downregulation of LINC01355 significantly restrained CD8+ T cell apoptosis, induced CD8+ T cell percentage, and enhanced the cytolysis activity when cocultured with OSCC cells. It has been reported that the Notch pathway represses CD8+ T cell activity in cancer patients. In our present study, we displayed that lack of LINC01355 suppressed OSCC malignant behaviors and enhanced the antitumor activity of CD8+ T cells via inactivating Notch signaling. We showed that decreased LINC01355 significantly restrained the Notch signal via a decrease of Notch-1, JAG-1, and HES-1. Repression of Notch1 reversed the effect of LINC01355 in OSCC cells. In conclusion, it was implied that LINC01355 might induce the development of OSCC via modulating the Notch signal pathway, which could provide a candidate therapeutic target for OSCC.

METTL3-mediated m6A mRNA modification promotes esophageal cancer initiation and progression via Notch signaling pathway

Esophageal cancer is a lethal malignancy with a high mortality rate, while the molecular mechanisms underlying esophageal cancer pathogenesis are still poorly understood. Here, we found that the N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) is significantly upregulated in esophageal squamous cell carcinoma (ESCC) and associated with poor patient prognosis. Depletion of METTL3 results in decreased ESCC growth and progression in vitro and in vivo. We further established ESCC initiation and progression models using Mettl3 conditional knockout mouse and revealed that METTL3-mediated m6A modification promotes ESCC initiation and progression in vivo. Moreover, using METTL3 overexpression ESCC cell model and Mettl3 conditional knockin mouse model, we demonstrated the critical function of METTL3 in promoting ESCC tumorigenesis in vitro and in vivo. Mechanistically, METTL3-catalyzed m6A modification promotes NOTCH1 expression and the activation of the Notch signaling pathway. Forced activation of Notch signaling pathway successfully rescues the growth, migration, and invasion capacities of METTL3-depleted ESCC cells. Our data uncovered important mechanistical insights underlying ESCC tumorigenesis and provided molecular basis for the development of novel strategies for ESCC diagnosis and treatment.

Doublecortin-Like Kinase 1 (DCLK1) Is a Novel NOTCH Pathway Signaling Regulator in Head and Neck Squamous Cell Carcinoma.

Despite recent advancements, the 5 year survival of head and neck squamous cell carcinoma (HNSCC) hovers at 60%. DCLK1 has been shown to regulate epithelial-to-mesenchymal transition as well as serving as a cancer stem cell marker in colon, pancreatic and renal cancer. Although it was reported that DCLK1 is associated with poor prognosis in oropharyngeal cancers, very little is known about the molecular characterization of DCLK1 in HNSCC. In this study, we performed a comprehensive transcriptome-based computational analysis on hundreds of HNSCC patients from TCGA and GEO databases, and found that DCLK1 expression positively correlates with NOTCH signaling pathway activation. Since NOTCH signaling has a recognized role in HNSCC tumorigenesis, we next performed a series of in vitro experiments in a collection of HNSCC cell lines to investigate the role of DCLK1 in NOTCH pathway regulation. Our analyses revealed that DCLK1 inhibition, using either a pharmacological inhibitor or siRNA, resulted in substantially decreased proliferation, invasion, migration, and colony formation. Furthermore, these effects paralleled downregulation of active NOTCH1, and its downstream effectors, HEY1, HES1 and HES5, whereas overexpression of DCLK1 in normal keratinocytes, lead to an upregulation of NOTCH signaling associated with increased proliferation. Analysis of 233 primary and 40 recurrent HNSCC cancer biopsies revealed that high DCLK1 expression was associated with poor prognosis and showed a trend towards higher active NOTCH1 expression in tumors with elevated DCLK1. Our results demonstrate the novel role of DCLK1 as a regulator of NOTCH signaling network and suggest its potential as a therapeutic target in HNSCC.

Notch Signaling Pathway Is Activated by Sulfate Reducing Bacteria.

Sulfate Reducing Bacteria (SRB), usually rare residents of the gut, are often found in increased numbers (called a SRB bloom) in inflammatory conditions such as Inflammatory Bowel Disease (IBD), pouchitis, and periodontitis. However, the underlying mechanisms of this association remain largely unknown. Notch signaling, a conserved cell-cell communication pathway, is usually involved in tissue development and differentiation. Dysregulated Notch signaling is observed in inflammatory conditions such as IBD. Lipolysaccharide and pathogens also activate Notch pathway in macrophages. In this study, we tested whether Desulfovibrio, the most dominant SRB genus in the gut, may activate Notch signaling. RAW 264.7 macrophages were infected with Desulfovibrio vulgaris (DSV) and analyzed for the expression of Notch signaling pathway-related proteins. We found that DSV induced protein expression of Notch1 receptor, Notch intracellular domain (NICD) and p21, a downstream Notch target, in a dose-and time-dependent manner. DSV also induced the expression of pro-IL1β, a precursor of IL-1β, and SOCS3, a regulator of cytokine signaling. The gamma secretase inhibitor DAPT or Notch siRNA dampened DSV-induced Notch-related protein expression as well the expression of pro-IL1β and SOCS3. Induction of Notch-related proteins by DSV was not affected by TLR4 -IN -C34(C34), a TLR4 receptor antagonist. Additionally, cell-free supernatant of DSV-infected macrophages induced NICD expression in uninfected macrophages. DSV also activated Notch pathway in the human epithelial cell line HCT116 and in mouse small intestine. Thus, our study uncovers a novel mechanism by which SRB interact with host cells by activating Notch signaling pathway. Our study lays a framework for examining whether the Notch pathway induced by SRB contributes to inflammation in conditions associated with SRB bloom and whether it can be targeted as a therapeutic approach to treat these conditions.

Predictive and Prognostic Biomarker Identification in a Large Cohort of Androgen Receptor-Positive Salivary Duct Carcinoma Patients Scheduled for Combined Androgen Blockade.

Simple SummaryAndrogen receptor signaling seems pivotal in aggressive salivary duct carcinoma. However, androgen deprivation therapy (ADT) frequently fails, emphasizing the need for biomarkers to predict treatment response. Here, the activities of several possible tumor-driving pathways were quantified and related to clinical outcome in a large cohort of SDC patients. Our results indicated that AR pathway activity and SRD5A1 expression levels can be used to predict response to ADT, which could prevent overtreatment in clinical practice.Patients suffering from recurrent or metastatic (R/M) salivary duct carcinoma (SDC) are often treated with combined androgen blockade (CAB). However, CAB frequently fails, resulting in a worse prognosis. Therefore, biomarkers that can predict treatment failure are urgently needed. mRNA from 76 R/M androgen receptor (AR)-positive SDC patients treated with leuprorelin acetate combined with bicalutamide was extracted from pre-treatment tumor specimens. AR, Notch, MAPK, TGFβ, estrogen receptor (ER), Hedgehog (HH), and PI3K signaling pathway activity scores (PAS) were determined based on the expression levels of target genes. Additionally, 5-alpha reductase type 1 (SRD5A1) expression was determined. These markers were related to clinical benefit (complete/partial response or stable disease ≥6 months) and progression-free and overall survival (PFS/OS). SRD5A1 expression had the highest general predictive value for clinical benefit and positive predictive value (PPV: 85.7%). AR PAS had the highest negative predictive value (NPV: 93.3%). The fitting of a multivariable model led to the identification of SRD5A1, TGFβ, and Notch PAS as the most predictive combination. High AR, high Notch, high ER, low HH PAS, and high SRD5A1 expression were also of prognostic importance regarding PFS and SRD5A1 expression levels for OS. AR, Notch PAS, and SRD5A1 expression have the potential to predict the clinical benefit of CAB treatment in SDC patients. SRD5A1 expression can identify patients that will and AR PAS patients that will not experience clinical benefit (85.7% and 93.3% for PPV and NPV, respectively). The predictive potential of SRD5A1 expression forms a rational basis for including SRD5A1-inhibitors in SDC patients’ treatment.

Abstract 174: In-depth transcriptomic comparisons provide novel insights into cancer metastasis

Abstract Comparison of the transcriptome between primary and metastatic cancer is frequently performed to study cancer metastasis; however, distilling plausible biology from the comparison results remains a big challenge. We show that in the differential expression (DE) analysis performed on bulk RNAseq data a large proportion of DE genes are identified due to technical issues instead of biological differences between primary and metastatic cancer cells. Next, we develop a computational pipeline DEBoost to minimize such artifacts and apply it to five cancer types. We perform comprehensive analysis on the retained tumor-intrinsic DE genes to explore the biological mechanisms underlying the metastasis of different cancer types. Many of the DE genes are associated with the progression of primary cancer, suggesting the differential expression may have already been established before metastasis. Clustering analysis on DE genes identifies CNVs that account for the transcriptomic change and we further confirm 19p13.12 amplification drives the metastasis of primary Basal-like breast cancer via activating NOTCH signaling pathway. We also find that cell cycling is more active in metastatic prostate cancer than in primary cancer, suggesting the difference of responses to treatment like cell cycling inhibitors. Finally, we show that some metastatic cancer cells tend to express genes whose expression is normally observed at the metastasis site and we experimentally validate this phenomenon in colorectal cancer liver metastasis. Our pan-cancer liver metastasis study provides novel biological insights into cancer metastasis and meanwhile demonstrates the feasibility of characterizing tumor-intrinsic DE genes in cancer metastasis using our computational pipeline. Citation Format: KE LIU, Benjamin S. Glicksberg, Shreya Paithankar, Bin Chen. In-depth transcriptomic comparisons provide novel insights into cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 174.

Down-regulation of EGFL8 regulates migration, invasion and apoptosis of hepatocellular carcinoma through activating Notch signaling pathway

BackgroundOur previous studies have reported the down-regulation of EGFL8 correlates to the development and prognosis of colorectal and gastric cancer. The present study is carried out to explore the expression pattern and role of EGFL8 in hepatocellular carcinoma (HCC).Methods and materialsEGFL8 expression in 102 cases of HCC tissues matched with adjacent non-tumorous liver tissues, a normal liver cell line and three liver cancer cell lines with different metastatic capacity was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. Moreover, the clinicopathological features and prognosis of HCC patients were correlated with expression of EGFL8. Subsequently, the gain-and loss-of-function experiments were carried out to investigate the biological function of EGFL8 in HCC. We also used N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)- phenylglycine t-butyl ester (DAPT), an inhibitor for Notch signaling pathway, in these experiments to verify the involvement of Notch signaling pathway in the effects of EGFL8. Additionally, a mouse model was established to investigate the effect of EGFL8 on metastasis of HCC cells. The expression of Notch signaling pathway in HCC cells and xenograft mouse tumors were detected by Western blot and immunohistochemistory.ResultsThe expression of EGFL8 was significantly decreased in HCC tissues and cell lines and EGFL8 down-regulation correlated to multiple nodules, vein invasion, high TNM stage and poor prognosis of HCC. Interestingly, the expression levels of EGFL8 in three liver cancer cell lines were negatively associated with their metastatic capacity. In vitro and in vivo experiments indicated that EGFL8 obviously suppressed metastasis and invasion of HCC cells but slightly promoted apoptosis. Meanwhile, the expression of Notch signaling pathway was obviously suppressed in EGFL8 overexpressed HCCLM3 cells and xenograft mouse tumors generated from these cells but markedly elevated in EGFL8 depleted Hep3B cells. Furthermore, the up-regulated expression of Notch signaling pathway and effects induced by EGFL8 knockdown in Hep3B cells could be counteracted by DAPT treatment.ConclusionThe down-regulation of EGFL8 was correlated to progression and poor prognosis of HCC and regulates HCC cell migration, invasion and apoptosis through activating the Notch signaling pathway, suggesting EGFL8 as a novel therapeutic target and a potential prognostic marker for HCC.

LncRNA HOXA-AS2 Activates the Notch Pathway to Promote Cervical Cancer Cell Proliferation and Migration

Long non-coding RNAs (lncRNAs) are crucial participants in cancer development. HOXA cluster antisense RNA 2 (HOXA-AS2) plays a tumor promoter role in bladder cancer. However, the functional role of HOXA-AS2 in cervical cancer remains unclear. Our study first found that HOXA-AS2 expression was up-regulated in cervical cancer cells. Then functional analysis including cell counting kit-8 (CCK-8), colony formation, transwell, and wound healing uncovered that reduction of HOXA-AS2 remarkably impeded cell proliferation and migration in cervical cancer. Additionally, luciferase reporter assays were performed to confirm that HOXA-AS2 activated Notch signaling pathway via the mediation of independent recombination signal binding protein for JK (RBP-JK) activity. As we know, Notch intracellular domain (NICD) is associated with RBP-JK in the nucleus to promote target genes in the Notch pathway. Through RNA immunoprecipitation (RIP), RNA pull down, and fluorescent in situ hybridization (FISH) assays, we observed that HOXA-AS2 combined with NICD. Moreover, the data from Co-IP assays indicated that HOXA-AS2 reduction weakened the interaction of NICD and RBP-JK. Collectively, HOXA-AS2 played a cancer-promoting role in cervical cancer development by modulating the Notch pathway, which might become a novel target for cervical cancer treatment.

Effects and mechanisms of polycaprolactone-cellulose acetate nanofiber scaffold loaded with rat epidermal stem cells on wound healing of full-thickness skin defects in rats

Objective: To explore the effects and mechanisms of polycaprolactone-cellulose acetate (PCL-CA) nanofiber scaffold loaded with rat epidermal stem cells (ESCs) on wound healing of full-thickness skin defects in rats. Methods: The experiment research method was applied. The primary ESCs were isolated from 1-3 d old Sprague-Dawley (SD) rats (undefined gender) by rapid adherent method and cultured by rapid adherent method. ESCs of the first passage were used for the subsequent experiments after the positive expressions of integrin β1 and cytokeratin 19 (CK19) in primary cells were identified respectively by flow cytometey and immunofluorescence method. PCL-CA nanofiber scaffolds with polycaprolactone and cellulose acetate as components were prepared by electrospinning technique. The topological structure of the nanofiber scaffolds was determined and the diameter of 25 fibers was measured by scanning electron microscope. The constructed PCL-CA nanofiber scaffolds were used as the culture substrate for ESCs, which were cultured in keratinocytes (KCs) medium to construct ESCs-nanofiber scaffold complex (hereinafter referred to as ESCs scaffold). After 3 days of culture, the morphology of ESCs in the scaffold and their relationship was observed by scanning electron microscopy. The ESCs in ESCs scaffold were set as PCL-CA nanofiber scaffold group, and the ESCs cultured with KCs medium in culture dishes coated with type Ⅳ collagen were set as type Ⅳ collagen group. Western blotting was used to detect the protein expression level of CK19 in ESCs in the two groups after 3 days of culture (n=3). The protein expressions of CK19 and proliferating nuclear antigen (PCNA) in ESCs in the two groups were detected by immunofluorescence method after 7 days of culture. A circular full-thickness skin wound of about 2 cm in diameter was prepared on both left and right sides of the back of 15 male SD rats aged 6-8 weeks. The rats were then equally divided into blank control group without implantation, scaffold alone group implanted with PCL-CA nanofiber scaffold, and ESCs scaffold group implanted with ESCs scaffold which were constructed after 3 days of culture according to the random number table. The percentage of wound areas on post injury day (PID) 3, 7, 14, and 21 was calculated (n=5). The new skin tissue at the wound edge was collected on PID 21, the wound healing quality was evaluated by Masson staining, and the protein expression levels of Notch1, Jagged1, and Hes1, which are key proteins of Notch signaling pathway, were detected by Western blotting (n=3). Data were statistically analyzed with one-way analysis of variance, one-way analysis of variance, analysis of variance for repeated measurement, independent sample t test, and Bonferroni correction. Results: The constructed PCL-CA nanofiber scaffolds had a porous, mesh-like, and multilayered three-dimensional structure, in which the surface of the fibers was smooth and non-porous, and the fiber diameter was (383±24) nm. The ESCs in ESCs scaffold showed intact cellular structures and were tightly attached to the scaffold after 3 days of culture. The cells were interconnected and fully extended on the surface of the scaffold to form a membrane. After 3 days of culture, the protein expression level of CK19 of ESCs in PCL-CA nanofiber scaffold group was significantly higher than that in type Ⅳ collagen group (t=24.56, P 0.05). On PID 21, the layer of new skin at the wound edge of rats in each group was intact; compared with that in blank control group or scaffold alone group, the new skin tissue at the wound edge of rats in ESCs scaffold group had more orderly collagen arrangement; the scaffolds in the new skin at the wound edge of rats were completely degraded in ESCs scaffold group and scaffold alone group. On PID 21, the protein expression levels of Notch1, Jagged1, and Hes1 in the new skin tissue at the wound edge of rats in scaffold alone group were similar to those in blank control group (t=1.70, 1.94, 0.18, P>0.05), while the protein expression levels of Notch1, Jagged1, and Hes1 in the new skin tissue at the wound edge of rats in ESCs scaffold group were significantly higher than those in scaffold alone group (t=13.31, 22.07, 20.71, P<0.01). Conclusions: PCL-CA nanofiber scaffolds can inhibit the differentiation of ESCs of rats without affecting their proliferation in vitro. ESCs scaffolds constructed through using PCL-CA nanofiber scaffolds as the carrier to culture ESCs of rats can significantly promote the wound healing of full-thickness skin defects in rats, and the mechanism may be related to the activation of Notch signaling pathway.

Clinical disease course and survival outcomes following disease recurrence in adenoid cystic carcinoma (ACC) with NOTCH signaling pathway activation.

6072 Background: ACC is a rare salivary cancer for which effective drug therapies remain lacking. The highest rates of disease recurrence are in patients with NOTCH pathway activation, which is reported in 10-20% of ACC tumors. Novel drugs targeting NOTCH signaling are under investigation in the recurrent and metastatic setting. To understand their clinical utility, there is an urgent need to better characterize the disease course and outcomes following current standard of care treatment from diagnosis and following recurrence. Methods: 120 patients with ACC underwent clinical review at a single UK Cancer Centre from 2017-19. Patients were retrospectively assessed for tumor NOTCH pathway activation using next generation sequencing (NGS) targeting NOTCH1/2/3 genes (n = 98) and/or by immunohistochemistry (IHC) for the NOTCH1 intra-cellular domain (NICD1) (n = 87). To understand the disease course with NOTCH pathway activation, treatment data including surgery, radiotherapy and systemic therapies were extracted and presented as swimmer plots. Kaplan-Meier survival analysis was performed and a difference in survival with/without NOTCH activation was calculated with log rank test. Overall survival (OS) was calculated both from diagnosis and from first confirmed disease recurrence or metastasis, and recurrence free survival (RFS) calculated from diagnosis. Results: Of 120 patients, median age was 46 years (22-74 years). 114/120 patients (95%) had confirmed disease recurrence at clinical review. The primary site was major salivary gland in 58/120 (48%), the others were minor salivary. NOTCH1/3 activating somatic mutations were identified in 11% by NGS (11/98) and NICD1 diffuse nuclear staining was seen in 6% by IHC (5/87) for overall NOTCH activation in 11% (13/120). In NOTCH activated ACC, primary site was major salivary gland in 7/13 (54%), and non-pulmonary visceral/bone metastases were present in 6/13 (46%). Consistent with other reports, patients with NOTCH activation (n = 13) had shorter RFS (0.9 vs 3.6 years, p = 0.11) and significantly reduced OS from diagnosis (4.0 vs 16.3 years, p &lt; 0.0001). Critically, as therapies targeting NOTCH signaling are being evaluated in recurrent/metastatic ACC, there was significantly reduced OS from time of first confirmed disease recurrence or metastasis (1.5 vs 9.6 years, p &lt; 0.0001). This reduction in OS for NOTCH activation following recurrence was seen consistently whether patients were classified using NGS (1.9 vs 9.6 years, p = 0.0009) or NICD1 IHC (0.8 vs 8.5 years, p &lt; 0.0001). Conclusions: This is the first study to report clinical outcomes for patients with NOTCH pathway activated ACC following disease recurrence. Although ACC is frequently considered an indolent disease, the short survival in this sub-group of ACC patients demonstrates the urgent need to develop effective drug therapies in this setting.

FZD2 promotes TGF-\u03b2-induced epithelial-to-mesenchymal transition in breast cancer via activating notch signaling pathway

BackgroundBreast cancer (BC) is one of the commonest female cancers, which is characterized with high incidence. Although treatments have been improved, the prognosis of BC patients in advanced stages remains unsatisfactory. Thus, exploration of the molecular mechanisms underneath BC progression is necessary to find novel therapeutic methods. Frizzled class receptor 2 (FZD2) belongs to Frizzled family, which has been proven to promote cell growth and invasion in various human cancers. The purpose of our current study was to detect the functions of FZD2 in BC and explore its underlying molecular mechanism.MethodsThe level of FZD2 was measured in BC tissues by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, immunohistochemistry (IHC), respectively. Cell Counting Kit-8 (CCK-8), colony formation assay, transwell assays, wound healing assay and flow cytometry analyses were separately conducted to detect cell viability, invasion, migration, apoptosis and cell cycle distribution. The levels of Epithelial-mesenchymal transition (EMT) biomarkers were examined by using Immunofluorescence assay. Xenograft tumorigenicity assay was performed to assess the effect of FZD2 on tumor growth in vivo.ResultsFZD2 mRNA and protein expression was abundant in BC tissues. Moreover, high level of FZD2 had significant correlation with poor prognosis in BC patients. In vitro functional assays revealed that silencing of FZD2 had suppressive effects on BC cell growth, migration and invasion. Animal study further demonstrated that FZD2 silencing inhibited BC cell growth in vivo. In addition, FZD2 induced EMT process in BC cells in a transforming growth factor (TGF)-β1-dependent manner. Mechanistically, knockdown of FZD2 led to the inactivation of Notch signaling pathway.ConclusionFZD2 facilitates BC progression and promotes TGF-β1-inudced EMT process through activating Notch signaling pathway.

Relevance of Notch Signaling for Bone Metabolism and Regeneration.

Notch1-4 receptors and their signaling pathways are expressed in almost all organ systems and play a pivotal role in cell fate decision by coordinating cell proliferation, differentiation and apoptosis. Differential expression and activation of Notch signaling pathways has been observed in a variety of organs and tissues under physiological and pathological conditions. Bone tissue represents a dynamic system, which is constantly remodeled throughout life. In bone, Notch receptors have been shown to control remodeling and regeneration. Numerous functions have been assigned to Notch receptors and ligands, including osteoblast differentiation and matrix mineralization, osteoclast recruitment and cell fusion and osteoblast/osteoclast progenitor cell proliferation. The expression and function of Notch1-4 in the skeleton are distinct and closely depend on the temporal expression at different differentiation stages. This review addresses the current knowledge on Notch signaling in adult bone with emphasis on metabolism, bone regeneration and degenerative skeletal disorders, as well as congenital disorders associated with mutant Notch genes. Moreover, the crosstalk between Notch signaling and other important pathways involved in bone turnover, including Wnt/β-catenin, BMP and RANKL/OPG, are outlined.

Deficiency of Rbpj Leads to Defective Stress-Induced Hematopoietic Stem Cell Functions and Hif Mediated Activation of Non-canonical Notch Signaling Pathways.

Deregulated notch signaling has been associated with human pathobiology. However, functions of notch pathways in hematopoiesis remain incompletely understood. Here, we ablated canonical notch pathways, through genetic deletion of Rbpj, in hematopoietic stem cells (HSCs). Our data identified that loss of canonical notch results in normal adult HSC pool, at steady state conditions. However, HSC maintenance and functions in response to radiation-, chemotherapy-, and cytokine- induced stress were compromised in the absence of canonical notch. Rbpj deficient HSCs exhibit decreased proliferation rates and elevated expression of p57Kip2. Surprisingly, loss of Rbpj resulted in upregulation of key notch target genes and augmented binding of Hes1 to p57 and Gata2 promoters. Further molecular analyses identified an increase in notch activity, elevated expression and nuclear translocation of Hif proteins, and augmented binding of Hif1α to Hes1 promoter in the absence of Rbpj. These studies, for the first time, identify a previously unknown role for non-canonical notch signaling and establish a functional link between Hif and Notch pathways in hematopoiesis.

Longdan Xiegan Decoction alleviates experimental autoimmune uveitis in rats by inhibiting Notch signaling pathway activation and Th17 cell differentiation

This study aimed to investigate the dynamic effects of the traditional Chinese medicine compound Longdan Xiegan Decoction (LXD) on the inhibition of Notch signaling pathway activation and T helper (Th) cell differentiation in rats with experimental autoimmune uveitis (EAU). Based on a network pharmacology strategy, we conducted protein interaction network analysis to construct an active ingredient-disease treatment network. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were further used to screen out the possible signaling pathways regulated by LXD in the treatment of uveitis. In the subsequent functional studies, we established an EAU rat model and investigated the regulatory role of LXD in the Notch signaling pathway and Th cell differentiation in rats with EAU. Female Lewis rats were randomly divided into a normal control (NC) group, an EAU group, and an LXD group. After the induction of EAU, the ocular inflammation and pathological changes in the rats in each group were observed; for documentation, a scanning laser ophthalmoscope (SLO) was used to observe fundus inflammation on day 12 after immunization. Additionally, quantitative polymerase chain reaction (Q-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of Notch1, DLL4, IL-10 and IL-17A in the spleen, lymph nodes and ocular tissues of each group at 0, 6, 9, 12, 15 and 18 days after immunization. In addition, the dynamic frequencies of the CD4+, CD8+, Th17 and Treg cell subsets in the spleen, lymph nodes and ocular tissues were measured by flow cytometry. We found that the Notch signaling pathway was activated and the Th17 frequency was elevated in rats with EAU, leading to disrupted CD4+/CD8+ and Th17/Treg balance. The expression of Notch1, DLL4 and IL-17 mRNA and proteins in the EAU and LXD groups reached a peak on day 12, and then gradually decreased (all P < 0.05), and the ratios of the CD4+/CD8+ and Th17/Treg also peaked on day 12. However, after treatment with LXD, the expression of Notch1, DLL4 and IL-17 mRNA and proteins was significantly decreased (all P < 0.05), and the CD4+/CD8+ and Th17/Treg ratios significantly gradually returns to balance. LXD can efficiently inhibit Th17 cell differentiation, decrease inflammatory cytokine expression, and restore the CD4+/CD8+ and Th17/Treg balance by inhibiting the activation of the Notch signaling pathway in rats with EAU, thus effectively alleviating eye inflammation, protecting eye tissue structures, and positively regulating the immune state of the whole body and the intraocular microenvironment.