Hes1 Expression Research Articles

Neural Differentiation Dynamics Controlled by Multiple Feedback Loops in a Comprehensive Molecular Interaction Network

Mathematical model simulation is a useful method for understanding the complex behavior of a living system. The construction of mathematical models using comprehensive information is one of the techniques of model construction. Such a comprehensive knowledge-based network tends to become a large-scale network. As a result, the variation of analyses is limited to a particular kind of analysis because of the size and complexity of the model. To analyze a large-scale regulatory network of neural differentiation, we propose a contractive method that preserves the dynamic behavior of a large network. The method consists of the following two steps: comprehensive network building and network reduction. The reduction phase can extract network loop structures from a large-scale regulatory network, and the subnetworks were combined to preserve the dynamics of the original large-scale network. We confirmed that the extracted loop combination reproduced the known dynamics of HES1 and ASCL1 before and after differentiation, including oscillation and equilibrium of their concentrations. The model also reproduced the effects of the overexpression and knockdown of the Id2 gene. Our model suggests that the characteristic change in HES1 and ASCL1 expression in the large-scale regulatory network is controlled by a combination of four feedback loops, including a large loop, which has not been focused on. The model extracted by our method has the potential to reveal the critical mechanisms of neural differentiation. The method is applicable to other biological events.

MiR-135a regulates renal fibrosis in rats with diabetic kidney disease through the Notch pathway.

To explore the influence of micro ribonucleic acid (miR)-135a on the renal fibrosis in rats with diabetic kidney disease (DKD) through the Notch signaling pathway. A total of 30 male Wistar rats weighing 200-220 g were selected and randomly divided into Control group (n=10), diabetes mellitus (DM) group (n=10), and miR-13a inhibitor group (n=10). Streptozotocin (STZ) was intraperitoneally injected daily to establish the DM model in rats of both DM group and miR-135a group, while normal saline was given daily through intraperitoneal injection in rats of Control group. After 4 weeks, the rats in miR-135a inhibitor group were intraperitoneally injected with miR-135a inhibitor, and those in Control and DM groups were administrated with an equal amount of normal saline. Changes in the blood glucose (BG), glycated hemoglobin (GHb), serum creatinine (Scr), triglyceride (TG), and total cholesterol (TC) of rats were evaluated, and the pathological changes in the renal tissues of DM rats were observed via hematoxylin-eosin (HE) staining. Sirius red staining was performed to observe the changes in collagen fibers in the kidney of all groups of rats. The expressions of Notch and Hes1 in the renal tissues of rats in each group were detected using immunohistochemistry. Immunofluorescence assay was employed to detect the positive expression of Notch in the renal tissues of rats. The mRNA expressions of Notch and miR-135a were detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Finally, Western blotting was conducted to detect the protein expressions of Notch, Notch intracellular domain (NICD) and Hes1. Compared with Control group, rats in DM group had substantially raised levels of BG, GHb, Scr, TG, and TC (p<0.05). HE staining showed that the rats in Control group had renal tubular cells with normal morphology and well-defined structure, while those in DM group exhibited evident cavitation in the renal tubular epithelium. Sirius red staining results manifested that the red collagen fibers were evenly distributed with light staining in the glomeruli and renal tubules of rats in Control group. In contrast, the collagen fibers of the glomeruli and renal tubules of rats in DM group exhibited deep and evident red staining. Moreover, compared with DM group, rats in miR-135a inhibitor group had notably faded red staining in the glomeruli and renal tubules of rats, evenly distributed collagen and remarkably decreased fibrotic nodules. According to immunohistochemistry detection results, the protein levels of Notch and Hes1 in the renal tubulointerstitial cells and renal tubular epithelial cells of rats in DM group were markedly higher than those in Control group. Compared with those in DM group, their protein levels were remarkably lowered in miR-135a inhibitor group (p<0.05). Immunofluorescence assay results revealed that the protein level of Notch in the renal tissues of rats in DM group was considerably higher than that in Control group (p<0.05), while its protein level in miR-135a inhibitor group was significantly lower than that in DM group. According to qRT-PCR results, compared with those in Control group, mRNA expressions of Notch mRNA and miR-135a in the rat kidney tissues were substantially raised in DM group (p<0.05), and they were notably lowered in miR-13a inhibitor group compared with those in DM group (p<0.05). Finally, Western blotting results manifested that the protein levels of Notch, NIC, and Hes1 in the renal tissues of rats in DM group were considerably higher than those in Control group (p<0.05), and that their protein expression levels in miR-135a inhibitor group were markedly lower than those in DM group (p<0.05). Inhibition of miR-135a can reduce the renal fibrosis in DKD rats through the Notch pathway.

Effects of 6-Shogaol on Notch signaling pathway in colonic epithelial cells of ulcerative colitis mice

To observe the regulatory effect of 6-Shogaol on Notch signal pathway in colonic epithelial cells of mice with ulcerative colitis. Forty Kunming mice were randomly divided into normal group (n=10) and model group (n=30). The model of ulcerative colitis was induced by free drinking of 2% dextran sulfate sodium salt(DSS). After 15 days, the mice were divided into model group, 6-gingerenol group and positive control group with 10 mice in each group. Normal group and model group were treated with normal saline, 6-gingerenol group was treated with 6-Shogaol 100 mg/(kg·d), positive control group was treated with sulfasalazine 100 mg/(kg·d), for 20 days. The histopathological changes of colon were observed, and the expressions of Hes-1 and Math-1protein in colonic epithelial cells were detected by immunofluorescence double labeling method. The expressions of Notch-1, Hes-1 and Math-1 mRNA in colonic epithelial tissue were detected by RT-PCR. The expressions of Notch-1, Hes-1 and Math-1 protein in colonic epithelial tissue was detected by Western blot. Compared with the normal group, the expression of Notch-1 and Hes-1 protein and the relative expression of mRNA in colonic epithelium of model group were significantly increased (P＜0.01), while the relative expressions of Math-1 mRNA and protein were decreased significantly (P＜0.01). Compared with the model group, the expressions of Notch-1 and Hes-1 protein and the relative expression of mRNA in colonic epithelium of 6-Shogaol group and sulfasalazine group were decreased significantly(P＜0.01), while the relative expressions of Math-1 mRNA and protein were increased significantly(P＜0.01). 6-Shogaol can inhibit the over activation of Notch pathway and regulate the balance of differentiation between colonic epithelialabsorptive cell line and secretory cell line and repair damaged mucosal tissue.

Changes in the expression of Notch and Wnt signalling molecules in human endometrial cancer.

Endometrial cancer is the sixth most frequent type of cancer among women worldwide. Type I adenocarcinomas account for 80-85% of endometrial cancer cases and sometimes require more aggressive treatment than theremaining part of this group. Therefore, molecular markers to stratify adenocarcinomas are needed. In this study, we analysed Notch and Wnt signalling in human endometrial cancer cases to evaluate these pathway elements as potential biomarkers for type I endometrial cancer. Endometrial samples were obtained from 47 women undergoing surgery for stageI-IV endometrial cancer in theNational Cancer Institute (Vilnius, Lithuania) in 2015-2016. Theexpression at themRNA level of signalling molecules genes (NOTCH1, NOTCH2, NOTCH3, NOTCH4, JAG1, JAG2, DLL1, HES1, AXIN2 and CTNNB1) was analysed by thequantitative real-time polymerase chain reaction. Relative expression of NOTCH1, NOTCH4, HES1 and β-catenin proteins in endometrioid adenocarcinoma was evaluated by theWestern blot method. The expression level of Notch receptors, ligands, and thetarget gene, as well as CTNNB1 and AXIN2, was reduced in stage I endometrioid adenocarcinoma if compared to theadjacent non-tumour tissue. Theexpression of all receptors, ligands, and target molecules was reduced in adenocarcinomas of later stages. Thestatistically significant correlations between transcript amounts of Notch receptors and ligands were found. There was astatistically significant difference in the gene expression of Notch signalling pathway components between different tumour differentiation grade samples. Apositive correlation between mRNA and protein theexpression level of NOTCH1, NOTCH4, HES1 was determined in stage I samples. Analysis of 47 human endometrial cancer samples revealedareduction in thetranscript levels of Notch and Wnt signalling molecule compared to theadjacent non-tumour tissue. These results suggest tumour suppressor function of Notch and Wnt signalling in human endometrial cancer. More detailed research on these signalling pathways should reveal their importance as potential biomarkers.

Allyl isothiocyanate treatment alleviates chronic obstructive pulmonary disease through the Nrf2-Notch1 signaling and upregulation of MRP1

AimsChronic obstructive pulmonary disease (COPD) is a disease with high morbidity and mortality worldwide, which can cause serious social and economic burdens. Allyl isothiocyanate (AITC) is one of the most common natural isothiocyanates and has been shown to have anti-inflammatory and antioxidant biological activities. The purpose of this study was to investigate whether AITC regulated Multidrug resistance-associated protein 1 (MRP1), reactive oxide species (ROS) and reduced glutathione (GSH) levels via Nrf2 and Notch1 signaling pathways to treat COPD and whether there was an interaction between these two pathways. Main methodsLung function indexes and histopathological changes in mice were determined by lung function instrument and HE staining, respectively. The protein expression was analyzed using immunohistochemistry and Western blotting. The mRNA expression was measured by RT-PCR in human bronchial epithelial cell line 16HBE. The contents of ROS, GSH and GSSG were detected by kits in 16HBE cells. Key findingsThe protein expression of Notch1, Hes1, MRP1, Nrf2, and HO-1 in lung tissues of WT mice and untransfected cells were significantly down-regulated in COPD, then significantly ameliorated in treatment groups. The protein expression of MRP1, Notch1 and Hes1 in lung tissues of Nrf2−/− mice were markedly reduced. There was a significant reduction in expression of Nrf2, HO-1 and MRP1 in si-Notch1 transfected cells. Pretreatment with AITC markedly improved oxidative stress and GSH-redox disorder in COPD. SignificanceOur study demonstrates that there is a potential interaction between Nrf2 and Notch1 signaling pathways during treatment of COPD.

HES1 and HES4 have non-redundant roles downstream of Notch during early human T-cell development.

In both mouse and human, Notch1 activation is the main initial driver to induce T-cell development in hematopoietic progenitor cells. The initiation of this developmental process coincides with Notch1-dependent repression of differentiation towards other hematopoietic lineages. Although well described in mice, the role of the individual Notch1 target genes during these hematopoietic developmental choices is still unclear in human, particularly for HES4 since no orthologous gene is present in the mouse. Here, we investigated the functional capacity of the Notch1 target genes HES1 and HES4 to modulate human Notch1-dependent hematopoietic lineage decisions and their requirement during early T-cell development. We show that both genes are upregulated in a Notch-dependent manner during early T-cell development and that HES1 acts as a repressor of differentiation by maintaining a quiescent stem cell signature in CD34+ hematopoietic progenitor cells. While HES4 can also inhibit natural killer and myeloid cell development like HES1, it acts differently on the T- versus B-cell lineage choice. Surprisingly, HES4 is incapable of repressing B-cell development, the most sensitive hematopoietic lineage with respect to Notch-mediated repression. In contrast to HES1, HES4 promotes initiation of early T-cell development, but ectopic expression of HES4, or HES1 and HES4 combined, is insufficient to induce T-lineage differentiation. Importantly, knockdown of HES1 or HES4 significantly reduces human T-cell development. Overall, we show that the Notch1 target genes HES1 and HES4 have non-redundant roles during early human T-cell development which may relate to differences in mediating Notch-dependent human hematopoietic lineage decisions.

MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes.

MicroRNAs (miRNAs/miRs) are considered to serve essential roles in podocyte apoptosis, and to be critical in the development of diabetic nephropathy (DN). Activation of the Notch signaling pathway has been demonstrated to serve an important role in DN development; however, its regulatory mechanisms are not fully understood. The present study used a high glucose (HG)-induced in vitro apoptosis model using mouse podocytes. Expression levels of miR-145-5p and its target, Notch1, and other key factors involved in the apoptosis signaling pathway were detected and measured by reverse transcription-quantitative PCR and western blotting. A luciferase reporter assay was performed to elucidate the miRNA-target interactions. The functions of miR-145-5p in apoptosis were detected using flow cytometry and TUNEL staining. The present study demonstrated that in HG conditions, miR-145-5p overexpression inhibited Notch1, Notch intracellular domain, Hes1 and Hey1 expression at the mRNA and protein levels. Notch1 was identified as a direct target of miR-145-5p. Furthermore, cleaved caspase-3, Bcl-2 and Bax levels were reduced significantly by miR-145-5p overexpression. These results indicate that miR-145-5p overexpression inhibited the Notch signaling pathway and podocyte lesions induced by HG. In conclusion, the results of the present study suggested that miR-145-5p may be a regulator of DN. Additionally, miR-145-5p inhibited HG-induced apoptosis by directly targeting Notch1 and dysregulating apoptotic factors, including cleaved caspase-3, Bcl-2 and Bax. The results of the present study provided evidence that miR-145-5p may offer a novel approach for the treatment of DN.

Modulation of the Notch System in Response to Wnt Inhibition Induces Restoration of the Rat Luteal Function.

The aim of this study was to investigate whether the Notch pathway is modulated in response to the downregulation of the Wnt/Β-catenin system in corpora lutea (CLs) from superovulated rats. To this end, we analyzed the effect of in vitro CL Wnt/Β-catenin inhibition on the expression of Notch members and on luteal function. Mechanically isolated rat CLs were cultured with ICG-001, a Wnt/B-catenin inhibitor. In this system, Wnt/B-catenin inhibition reduced progesterone production and decreased StAR protein levels. Besides, Wnt/B-catenin inhibition stimulated the Notch system, evidenced by an increase in Hes1 expression, and promoted the expression of selected Notch family members. At long incubation times, StAR levels and progesterone concentration reached the control values, effects probably mediated by the Notch pathway. These results provide the first evidence of a compensatory mechanism between Wnt/B-catenin signaling and the Notch system, which contributes to the homeostasis of luteal cells.

Inhibition of HES-1 might play a protective role in endothelial cells under cholesterol stimulation via PI3K/AKT signaling pathway.

The occurrence of atherosclerotic cardiovascular disease (ASCVD) was closely related to low-density lipoprotein (LDL) cholesterol. HES-1 is critical for maintains of stem cells, quiescent cells or cancer cells, and contributes to drug resistance and metastasis of tumor cells. In this study, we established a cell model of HES-1 inhibition and overexpression in Ea.hy 926 cells, and firstly detected the proliferation rete of Ea.hy 926 cells under cholesterol stimulation using MTT assay, and apoptosis of Ea.hy 926 cells were detected using flow cytometry. Expression of HES-1, apoptosis related proteins and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway were detected using Western blotting analysis. The expression of apoptotis related genes were detected using polymerase chain reaction (PCR) method. The concentration of angiogenesis cytokines was detected using enzyme-linked immunosorbent assay (ELISA) method. We found that proliferation of Ea.hy 926 cells was inhibited after stimulation of cholesterol, inhibition of HES-1 expression would reduce this effect. We also found that expression of apoptosis related molecules was increased and expressions of angiogenesis factors were decreased after cholesterol treatment. Besides, we revealed that these effects were mediated via PI3K/AKT signaling pathway, and HES-1 inhibition could increase the activity of this signaling pathway.

Oscillatory expression of Hes1 regulates cell proliferation and neuronal differentiation in the embryonic brain.

The expression of the transcriptional repressor Hes1 oscillates in many cell types, including neural progenitor cells (NPCs), but the significance of Hes1 oscillations in development is not fully understood. To examine the effect of altered oscillatory dynamics of Hes1, we generated two types of Hes1 knock-in mice, a shortened (type-1) and an elongated (type-2) Hes1 gene, and examined their phenotypes focusing on neural development. Although both mutations affected Hes1 oscillations, the type-1 mutation dampened Hes1 oscillations more severely, resulting in much lower amplitudes. The average levels of Hes1 expression in type-1 mutant NPCs were also lower than in wild-type NPCs but similar to or slightly higher than those in Hes1 heterozygous mutant mice, which exhibit no apparent defects. Whereas type-2 mutant mice were apparently normal, type-1 mutant mice displayed smaller brains than wild-type mice and upregulated proneural gene expression. Furthermore, proliferation of NPCs decreased and cell death increased in type-1 mutant embryos. When Hes3 and Hes5 were additionally deleted, neuronal differentiation was also accelerated, leading to microcephaly. Thus, robust Hes1 oscillations are required for maintenance and proliferation of NPCs and the normal timing of neurogenesis, thereby regulating brain morphogenesis.

GRWD1 promotes cell proliferation and migration in non-small cell lung cancer by activating the Notch pathway

GRWD1 is a member of the WD repeat protein family that is over-expressed in various cancer cell lines and associated with poor prognosis in patients with cancer. However, its biological function and mechanism in non-small cell lung cancer (NSCLC) remain unclear. In this study, we aimed to elucidate the role of GRWD1 in NSCLC. Immunohistochemistry on tumor specimens from 170 patients showed that GRWD1 is highly expressed in NSCLC tissues and positively correlated with tumor size, lymph node metastasis, and P-TNM stage, but negatively correlated with differentiation and prognosis. We found that GRWD1 promotes cell colony formation by affecting the expression of Cyclin B1, CDK1, and p27 and inducing G2/M transition. GRWD1 was also found to stimulate cell migration through RhoA, RhoC, and CDC42, and induce epithelial–mesenchymal transition by affecting the expression of E-cadherin, N-cadherin, Vimentin, Snail, Zeb1, and ZO-1. Our results indicated that the GRWD1 can activate the Notch signaling pathway by affecting the Notch intracellular domain and promoting the expression of Hes1. Our use of DAPT to suppress Notch signaling confirmed that GRWD1 promotes the progression of NSCLC through the Notch signaling pathway and may be a potential prognostic biomarker and therapeutic target for this disease.

Tong-Xie-Yao-Fang improves intestinal permeability in diarrhoea-predominant irritable bowel syndrome rats by inhibiting the NF-\u03baB and notch signalling pathways

BackgroundTong-Xie-Yao-Fang (TXYF) has been shown to be effective in diarrhoea-predominant irritable bowel syndrome (IBS-D) patients. However, the underlying mechanism remains to be clarified. The aim of this study was to investigate the efficacy and related mechanisms of TXYF in an IBS-D rat model.MethodsThe IBS-D rat model was established with 4% acetic acid and evaluated by haematoxylin-eosin (HE) staining. Then, IBS-D rats were divided into control, TXYF and rifaximin groups and treated intragastrically with normal saline, TXYF and rifaximin, respectively, for 14 days. The following indicators were measured before and after treatment: defecation frequency, faecal water content (FWC) and colorectal distension (CRD). Histopathological changes in the distal colon were observed after treatment. The expression of OCLN and ZO1 in the distal colon of IBS-D rats reflected the intestinal mucosal permeability, as measured by qRT-PCR, western blot, and enzyme-linked immunosorbent assays (ELISAs). The NF-κB and Notch signalling pathways and inflammation-related factors were investigated.ResultsAfter treatment with TXYF, the defecation frequency, FWC and CRD were significantly lower than those in the model group (P < 0.05). HE staining showed that colonic epithelial cells (CECs) in the IBS-D rats displayed significant oedema, impaired intestinal mucosal integrity and an increased influx of inflammatory cells. A significant reduction in granulocyte and CEC oedema was observed after the administration of TXYF and rifaximin compared to that of the model group and blank group (P < 0.05). TXYF significantly upregulated the expression of OCLN and ZO-1 and downregulated inflammation-related factors (IL-6, IL-1β, and TNF-α and the chemokine KC) in IBS-D rats compared to those in the model group rats (P < 0.05). In terms of the NF-κB and Notch signalling pathways, the expression of NICD, p-ERK, Hes-1 and p-P65 decreased significantly in the TXYF and rifaximin groups, while the expression of ATOH1 increased significantly compared to that in the model group (P < 0.05).ConclusionTXYF can effectively improve intestinal permeability and enhance intestinal mucosal barrier function, which may be related to inhibition of the inflammatory cascade and the NF-κB and Notch signalling pathways.

Allyl isothiocyanate upregulates MRP1 expression through Notch1 signaling in human bronchial epithelial cells.

Multidrug resistance associated protein-1 (MRP1) and Notch signaling are closely related and both play a critical role in chronic obstructive pulmonary disease (COPD) establishment and progression. The aim of our work was to test whether Notch1 is involved in allyl isothiocyanate (AITC) induced MRP1 expression. We used cigarette smoke extract (CSE) to simulate the smoking microenvironment in vitro. The results demonstrated that CSE led to apoptosis as well as reduced the expression of Notch1, Hes1, and MRP1, while AITC significantly reversed this downregulation. Transfected with Notch1 siRNA downregulated MRP1 expression and activity, aggravated the suppression effect by CSE, and abolished the AITC-induced Notch1, Hes1, and MRP1 expression. Validation of the correlation between Notch1 and MRP1 was implemented by gel-shift assays (electrophoretic mobility shift assay). The result revealed an interaction between a specific promoter region of MRP1 and the intracellular domain of Notch1. In conclusion, Notch1 signaling positively regulated MRP1 in 16HBE cells and AITC induced MRP1 expression and function may be attributed to Notch1 signaling. These findings show that Notch1 and MRP1 might have a potential protective effect in the COPD process and become a new therapeutic target for COPD or other lung diseases. It also provides a theoretical basis for the therapeutic effects of AITC.

γ分泌酶抑制剂阻断Notch-Hes1信号通路对银屑病样皮炎小鼠γδT17细胞表达的影响

Objective To evaluate the effect of Notch-Hes1 signaling blockade by a γ-secretase inhibitor on the expression of γδT17 cells in a mouse model of psoriasis-like skin inflammation. Methods Forty-five healthy specific pathogen-free (SPF) male BALB/c mice were randomly divided into control group, model group and intervention group by simple random sampling. The model group and intervention group were both topically treated with imiquimod 5% cream (62.5 mg once a day) on the shaved back, the intervention group were then intraperitoneally injected with the γ-secretase inhibitor DAPT (10 mg/kg once a day) immediately after topical application of imiquimod, and the control group were topically treated with equivalent amount of vaseline once a day. After 6-day treatment, psoriasis area and severity index (PASI) was used to evaluate changes of skin lesions. On day 7, blood samples were obtained from all the mice through heart puncture after anesthetization, and spleen and skin tissues were acquired to prepare single cell suspension. Spleen index was compared among the 3 groups. Skin tissues on the mouse back were resected and subjected to hematoxylin-eosin staining to observe histopathological changes. Flow cytometry was performed to determine the percentage of γδT17 cells in the spleen and skin tissues, real-time reverse transcription (RT) -PCR to measure the mRNA expression of Hes1 in single cell suspension of the spleen, and enzyme-linked immunosorbent assay (ELISA) to determine the serum level of interleukin (IL) -17A. Statistical analysis was carried out by using one-way analysis of variance and repeated measures analysis of variance for comparison of indices among groups, and Pearson correlation analysis for evaluating the correlation between different indices. Results Twenty-four hours after the final treatment, the intervention group showed milder psoriasis-like skin inflammation, lower PASI score, and milder degree of epidermal thickening and dermal inflammatory cell infiltration compared with the model group. The model group showed significantly increased spleen index (12.534 ± 1.636) , proportions of γδT17 cells in the spleen (24.659% ± 4.603%) and skin tissues (22.127% ± 5.670%) , mRNA expression of Hes1 in the spleen (4.867 ± 0.543) , and serum level of IL-17A ([22.478 ± 2.776] ng/L) compared with the control group (all P < 0.01) . However, the above indices were significantly lower in the intervention group (9.449 ± 1.040, 14.966% ± 5.770%, 13.631% ± 5.946%, 2.541 ± 0.347, [18.639 ± 1.816] ng/L) than in the model group (all P < 0.01) . In the model group and intervention group, there were positive correlations between the proportions of γδT17 cells in the spleen and serum levels of IL-17A (r = 0.56, 0.53 respectively, both P < 0.05) , between the proportions of γδT17 cells in skin lesions and PASI scores (r = 0.56, 0.52 respectively, both P < 0.05) , as well as between the mRNA expression of Hes1 in the spleen and the proportions of γδT17 cells (r = 0.61, 0.58 respectively, both P < 0.05) or serum levels of IL-17A (r = 0.60, 0.54 respectively, both P < 0.05) . Conclusion Notch-Hes1 signaling blockade by γ-secretase inhibitor can markedly inhibit the expression of γδT17 cells, and effectively alleviate the severity of psoriasis-like skin inflammation in mouse models. Key words: Psoriasis; Amyloid precursor protein secretases; Receptors, Notch; Th17 cells; Interleukin-17; Hes1; γδT17 cells; γ-Secretase inhibitor

EGFL7 Antagonizes NOTCH Signaling and Represents a Novel Therapeutic Target in Acute Myeloid Leukemia (AML)

Epidermal growth factor-like domain 7 (EGFL7) is a secreted protein. We previously found that EGFL7 is up-regulated in primary AML blasts and that a high mRNA expression correlates with shorter event-free and overall survival in AML patients (Papaioannou et al. PNAS 2017;114(23)). However, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown. To identify EGFL7 binding partners, we took an unbiased high-throughput approach by using an antibody interaction-array to measure the ability of EGFL7 to bind directly ~400 proteins expressed by primary AML blasts. Using this strategy, we found in cell lysates of 3 AML patients that EGFL7 binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH (in all patients P&lt;0.001 compared to control). The direct interaction between EGFL7 and NOTCH1 as well as NOTCH2 receptor was confirmed in co-immunoprecipitation assays in THP1 and primary patient cells. Next, we stimulated AML blasts from 3 patients with recombinant EGFL7 (rEGFL7) and found that it reduced NOTCH intracellular domain (NICD1/2, Figure 1A), indicating that EGFL7 inhibits NOTCH activation. In line with this, we found that stimulation of AML patients samples' with rEGFL7 resulted in a decreased expression of its well-known downstream target gene HES1 (Figure 1B; in all patients P&lt;0.05 compared to unstimulated (unstim) cells). On the other hand, treatment with an anti-EGFL7 blocking antibody (AB) caused increased levels of NICD1/2 (Figure 1C) and expression of HES1 (Figure 1D, in all patients P&lt;0.05 compared to cells treated with IgG) indicating a reactivation of NOTCH signaling. Moreover, the treatment of THP1 cells with the anti-EGFL7 blocking AB resulted in increased apoptosis (1.9 fold increase compared to IgG control, P&lt;0.05) and differentiation (14.9 fold increase in CD11B expression compared to IgG control, P&lt;0.05; 5.6 fold increase in CD14 expression compared to IgG control, P&lt;0.05). To determine whether blocking EGFL7 could provide a new targeted therapy for patients with AML, we treated 3 independent AML models (i.e. an AML cell line based xenotransplant model (n=4 mice per group), a primary murine AML model (n=7 mice per group), and a patient derived xenotransplant model (n=4 mice per group)) with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival in vivo. In all models, we demonstrated that in vivo treatment with anti-EGFL7 results in prolonged overall survival (cell line model: P&lt;0.01; primary murine AML model: P&lt;0.01; for the patient derived xenotransplant model: P&lt;0.05). In conclusion, our data demonstrate that EGFL7 contributes to NOTCH silencing in AML by antagonizing canonical NOTCH ligand binding. Reactivation of NOTCH signaling in vivo using anti-EGFL7 results in prolonged survival of leukemic mice, supporting EGFL7 might be a novel therapeutic target in AML. Figure 1 A AML patient blasts were cultured in the presence or absence (Unstim) of rEGFL7. Total proteins were extracted for immunoblotting with β-ACTIN as loading control. B Total RNA was extracted for quantitative real time (qRT-PCR) analysis of HES1 mRNA normalized to β-ACTIN control (*P&lt;0.05, **P&lt;0.01, ***P&lt;0.001) stimulated with +rEGFL7 vs Unstim. C Patient blasts were cultured in presence of 100 μg/ml of normal IgG or 100 μg/ml anti-EGFL7 antibody (@EGFL7) for 4 hours. Total protein was extracted for immunoblotting of NICD with b-ACTIN as loading control. D Patient blasts were cultured with 100 μg/ml of normal IgG or anti-EGFL7 antibody for 2 hours and 10 hours. Total RNA was extracted for qRT-PCR analysis of HES1 mRNA with b-ACTIN as internal control. *P&lt;0.05, ***P&lt;0.001 vs IgG control. E Treatment with anti-EGFL7 blocking antibody prolonged survival compared to IgG1 controls in 3 independent mouse models. Disclosures No relevant conflicts of interest to declare.

Frontline Treatment with Ibrutinib Plus Rituximab (IR) Followed By Short Course R-Hypercvad/MTX Is Extremely Potent and Safe in Patients (age ≤ 65 years) with Mantle Cell Lymphoma (MCL) - Results of Phase-II Window-1 Clinical Trial

Frontline Treatment with Ibrutinib Plus Rituximab (IR) Followed By Short Course R-Hypercvad/MTX Is Extremely Potent and Safe in Patients (age ≤ 65 years) with Mantle Cell Lymphoma (MCL) - Results of Phase-II Window-1 Clinical Trial

Prognostic genes of triple-negative breast cancer identified by weighted gene co-expression network analysis.

Triple-negative breast cancer (TNBC) is characterized by a deficiency in the estrogen receptor (ER), progesterone receptor (PR) and HER2/neu genes. Patients with TNBC have an increased likelihood of distant recurrence and mortality, compared with patients with other subtypes of breast cancer. The current study aimed to identify novel biomarkers for TNBC. Weighted gene co-expression network analysis (WGCNA) was applied to construct gene co-expression networks; these were used to explore the correlation between mRNA profiles and clinical data, thus identifying the most significant co-expression network associated with the American Joint Committee on Cancer-TNM stage of TNBC. Using RNAseq datasets from The Cancer Genome Atlas, downloaded from the University of California, Santa Cruz, WGCNA identified 23 modules via K-means clustering. The most significant module consisted of 248 genes, on which gene ontology analysis was subsequently performed. Differently Expressed Gene (DEG) analysis was then applied to determine the DEGs between normal and tumor tissues. A total of 42 genes were positioned in the overlap between DEGs and the most significant module. Following survival analysis, 5 genes [GIPC PDZ domain containing family member 1 (GIPC1), hes family bHLH transcription factor 6 (HES6), calmodulin-regulated spectrin-associated protein family member 3 (KIAA1543), myosin light chain kinase 2 (MYLK2) and peter pan homolog (PPAN)] were selected and their association with the American Joint Committee on Cancer-TNM diagnostic stage was investigated. The expression level of these genes in different pathological stages varied, but tended to increase in more advanced pathological stages. The expression of these 5 genes exhibited accurate capacity for the identification of tumor and normal tissues via receiver operating characteristic curve analysis. High expression of GIPC1, HES6, KIAA1543, MYLK2 and PPAN resulted in poor overall survival (OS) in patients with TNBC. In conclusion, via unsupervised clustering methods, a co-expressed gene network with high inter-connectivity was constructed, and 5 genes were identified as biomarkers for TNBC.

Activation of CREB-binding protein ameliorates spinal cord injury in tabersonine treatment by suppressing NLRP3/Notch signaling.

Spinal cord injury (SCI) alters the integrity of the spinal cord, which leads to loss of multiple organs' function including locomotor function. The present study evaluates the protective effect of tabersonine against SCI. SCI was induced by traumatic injury and animals were treated with tabersonine 20 and 40 mg/kg, intraperitoneally for the period of 10 days. Tabersonine's effect was determined by estimating locomotor and neurological function in spinal cord injured rats. Moreover, mediators of inflammation were estimated using enzyme-linked immunosorbent assay (ELISA) and the effect of tabersonine on Notch/inflammasome signaling was estimated by reverse transcription polymerase chain reaction (RT-PCR), western blot assay and immunohistochemistry. Apoptosis of neuronal cells was estimated by staining with Nissl stain on spinal cord tissue in SCI rats. Data of the study suggest that neurological and motor functions were improved in the tabersonine treated group compared to the spinal cord injured (SI) group. There was a decrease in the mediators of inflammation in the spinal cord tissue of the tabersonine treated group compared to the SI group. Treatment with tabersonine ameliorates the altered expression of NICD, Nestin and Hes-1 protein and mRNA expression of Notch-1 and Hes-1 in the SCI rats. It was also observed that the tabersonine treated group showed activation of CREB and inhibition of the NLRP-3 pathway in SCI rats. Moreover, apoptosis of neuronal cells was reduced in the tabersonine treated group compared to the SI group. Data of the investigation suggest that tabersonine protects against spinal cord injury by activating CREB and reducing NLRP3/Notch signaling.

143P - NOTCH3 expression predicts poor survival in advanced esophageal squamous cell cancers

BackgroundOesophageal cancer is the sixth leading cause of cancer-related mortality with more than 90% of oesophageal cancer being squamous cell carcinomas (ESCC) in Asian countries. Prognosis of ESCC remains extremely poor and identification of better prognostic factors is an unmet need. MethodsWe investigated the immune- histochemical expression of Notch1, Notch2, Notch 3, HES-1 (Hairy enhancer of Split -1) (a downstream transcription factor) and p21 among ESCC and non-tumour controls. Non-tumour controls consisted of diagnostic biopsies of GERD patients in which Barrett’s and erosive esophagitis had been ruled out. We also investigated the association of these markers with clinico-pathological features and survival. We included 46 patients of ESCC and 42 controls. IHC markers were performed according to the standard guidelines and analysed with a semi-quantitative H –score (The H score was calculated as: H score= D X I with values ranging from 1-15). ResultsThe median age was 60 (37-78 years). M:F ratio was 30: 16. 65.2 % were smokers and 19.6 % were tobacco chewers. 38 cases were locally advanced (Stage III) and 8 were metastatic at baseline. Palliative intent therapy was offered to 24 % and definitive therapy to 76%. An ECOG -PS of 1,2 and 3 was observed in 26, 16 and 4 patients respectively. At a median follow up of 50.67 months, the median overall survival was10.47 mon (range= 8.23-15.1) mon. Using a cut-off score of 7.5 to define positivity, NOTCH 1,2,3 as well as p21 were found to be significantly over-expressed in ESCC as compared to normal controls, while there was no difference in Hes1 expression. Location, grade, baseline metastases, duration of symptoms showed no association with NOTCH 1,2 or 3 positivity. On univariate analysis, male sex, age >60 years, smokers, ECOG-PS>1, Notch 3 H score was found significant. On multivariate analysis, smokers (HR = 3.53, p = 0.013), ECOG-PS>1 (HR = 2.88, p = 0.006) and Notch3 H score (HR = 2.38, p = 0.047) emerged as independent adverse prognostic factors. ConclusionsNOTCH 1,2 and 3 are highly expressed in ESCC as compared to controls. Notch3 H score is an independent predictor of survival in ESCC and merits evaluation in larger studies. Legal entity responsible for the studyRaja Pramanik. FundingIntramural Research Fund, Research Section, All India Institute of Medical Sciences, New Delhi. DisclosureAll authors have declared no conflicts of interest.

Genistein Protects Against Burn-Induced Myocardial Injury via Notch1-Mediated Suppression of Oxidative/Nitrative Stress.

Genistein (Gen) exhibits strong anti-oxidative/antinitrative activity and cardioprotective effects in several models; however, its role in burn-induced myocardial injury is unknown. This study investigated the protective effect of Gen on burn-induced myocardial injury and aimed to elucidate the mechanism of protection. Mice were injected with Gen, intraperitoneally, at different dose immediately after burn injury. The expression levels of Notch-1 intracellular domain (NICD1) and hairy and enhancer of split (Hes-1) were determined by immunoblotting. Conditional Notch-RBP-J knockout mice were used to investigate the mechanisms of Gen-induced cardioprotection. Gen alleviated burn-induced myocardial injury, as shown by improved left ventricle ejection fraction, decreased serum lactate dehydrogenase and creatine kinase levels, and apoptosis. Moreover, Gen decreased expressions of inducible nitric oxide (NO) synthase and gp, reduced NO and superoxide anions production, and ameliorated their cytotoxic reaction product, peroxynitrite. More importantly, Gen significantly up-regulated the expression of NICD1 and Hes1 after burn injury. In addition, genetic knockout of Notch1 not only blocked the cardioprotection of Gen but also markedly attenuated Gen-induced anti-oxidative/antinitrative effect. These results demonstrate, for the first time, that Gen treatment attenuates burn-induced myocardial injury via the Notch1 mediated suppression of oxidative/nitrative stress.