Notch Proteins Research Articles

Immunohistochemical expression of Neurogenic Locus Notch Homolog 4 (Notch4) in invasive breast carcinoma: Correlation with clinicopathological parameters and immunohistochemical-based subtypes

IntroductionStarting out with its discovery as small notches on fly wings, Neurogenic Locus Notch Homolog 4 (Notch4) signaling has been sparked as unique pathway implicated in cellular multiplication, differentiation, and regulation of stem cells. Its aberrant activation causes arrays of cancers including breast cancer. ObjectivesThe aim of the present study was to evaluate the immunohistochemical expression level of Notch4 and its subcellular localization in invasive breast carcinoma. The correlation between Notch4 expression and both of clinicopathological parameters and immunohistochemical-based subtypes of studied cases was also assessed. Methods and materialsImmunohistochemical expression of Notch4 receptor was examined in 60 specimens of paraffin-embedded sections of invasive breast cancer. Normal and hyperplastic breast tissue adjacent to carcinoma cells was also included in the study. ResultsThere was a significant increase in the expression level of Notch4 protein in breast cancer, compared to that of normal breast tissue and hyperplastic breast lesions. Also, there was a statistical significant correlation between Notch4 expression level and tumor size, tumor grade, nodal metastasis, lymphovascular invasion, human epidermal growth factor receptor 2 (her2) status, Her2-enriched and triple negative subtypes, and Ki67. Furthermore, an inverse significant correlation was found between Notch4 expression and both of age and estrogen receptor (ER). No statistical significant correlation was found between Notch4 expression and tumor histological subtypes, Tumor infiltrating lymphocytes (TILs), and fibrosis. ConclusionNotch4 overexpression has been implicated in breast cancer development, progression and emergence of aggressive biological phenotypes.

ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping

Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in mAb. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of noncollagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a nonreduced mAb.

Molecular docking analysis of phytocompounds from Andrographis paniculata binding with proteins in the notch-signaling pathway.

It is of interest to document the molecular docking analysis of phytocompounds from Andrographis paniculata binding with protein NOTCH1 in the Notch-signaling pathway in the context of cancer. Hence, we document the binding features of neoandrographolide, 14-deoxyandrographolide, androgapholide and andrograpanin with proteins in the notch-signaling pathway for further consideration.

Abstract 16626: Endothelial Zinc Transporter Zip8 Regulates Ventricular Trabeculation and Compaction

Trabeculation and subsequent compaction of the ventricular wall are essential for normal heart morphogenesis. In human, excessive trabeculation and impaired myocardial compaction during earlyheart development lead to left ventricular non compaction (LVNC), a third most common cardiomyopathies after dilated and hypertrophic cardiomyopathies. LVNC is also associated with cardiac arrhythmia and sudden cardiac death. The etiology of LVNC is poorly understood. Recently our group identified that Zip8, a Zinc transporter, implicated in ventricular trabeculation and compaction (JCI, 2018,PMID: 29337306). However, it remains elusive how Zip8 regulates these processes through cellular and molecular mechanisms. We conditionally ablated Zip8 in the endothelial cells, epicardial cells and cardiomyocytes in the developing heart respectively. Strikingly, deletion of Zip8 in the endothelial cellsgave rise to LVNC phenotypes, identical to cardiac phenotypes from the global Zip8 knockouts. Incontrast, hearts with deletions of Zip8 in either epicardium or myocardium developed normally. Further,we found only conditional deletion of Zip8 in the endothelium in the early developing hearts (at E8.5)generated LVNC but not in the late gestation stage (E13.5), suggesting that Zip8 plays critical roles during the initial trabeculation stage. Interestingly, we found that endocardial Zip8 deficiency resulted inthe upregulation of Notch signaling, as evidenced by the increase of intracellular domain of notch protein(NICD) and its downstream target gene, bone morphogenetic protein 10 (BMP10). When the notch signaling is inhibited in the endothelial Zip8 knockout hearts either by DAPI, a γ secretase inhibitor, or by genetic deletion of Rbpj, the LVNC cardiac phenotypes are partially rescued. These results suggest thatZip8 orchestrates myocardial trabeculation and compaction by regulating notch signaling.Collectively, our study provides novel insights into the mechanisms of LVNC, suggesting that genetic mutations in Zip8 or zinc deficiency may contribute to LNVC in humans.

CCN3 Signaling Is Differently Regulated in Placental Diseases Preeclampsia and Abnormally Invasive Placenta.

ObjectivesAn adequate development of the placenta includes trophoblast differentiation with the processes of trophoblast migration, invasion, cellular senescence and apoptosis which are all crucial to establishing a successful pregnancy. Altered placental development and function lead to placental diseases such as preeclampsia (PE) which is mainly characterized by insufficient trophoblast invasion and abnormally invasive placenta (AIP) disorders (Placenta accreta, increta, or percreta) which are characterized by excessive trophoblast invasion. Both of them will cause maternal and fetal morbidity/mortality. However, the etiology of these diseases is still unclear. Our previous study has shown that the matricellular protein nephroblastoma overexpressed (NOV, CCN3) induces G0/G1 cell cycle arrest, drives trophoblast cells into senescence and activates FAK and Akt kinases resulting in reduced cell proliferation and enhanced migration capability of the human trophoblast cell line SGHPL-5. The present study focuses on whether CCN3 can alter cell cycle-regulated pathways associated with trophoblast senescence and invasion activity in pathological versus gestational age-matched control placentas.MethodsCell cycle regulator proteins were investigated by immunoblotting and qPCR. For localization of CCN3, p16, p21, and Cyclin D1 proteins, co-immunohistochemistry was performed.ResultsIn early-onset PE placentas, CCN3 was expressed at a significantly lower level compared to gestational age-matched controls. The decrease of CCN3 level is associated with an increase in p53, Cyclin E1 and pRb protein expression, whereas the level of cleaved Notch-1, p21, Cyclin D1, pFAK, pAKT, and pmTOR protein decreased. In term AIP placentas, the expression of CCN3 was significantly increased compared to matched term controls. This increase was correlated to an increase in p53, p16, p21, Cyclin D1, cleaved Notch-1, pFAK, pAkt, and pmTOR whereas pRb was significantly decreased. However, in late PE and early AIP placentas, no significant differences in CCN3, p16, p21, Cyclin D1, p53, and cleaved Notch-1 expression were found when matched to appropriate controls.ConclusionsCCN3 expression levels are correlated to markers of cell cycle arrest oppositely in PE and AIP by activating the FAK/AKT pathway in AIP or down-regulating in PE. This may be one mechanism to explain the different pathological features of placental diseases, PE and AIP.

MicroRNA-27 Inhibits Autophagy and Promotes Proliferation of Multiple Myeloma Cells by Targeting the NEDD4/Notch1 Axis.

Multiple myeloma (MM) is a malignant tumor disease that seriously affects the health of patients. Previous studies have shown the crucial role of autophagy in the development of MM. Therefore, the study aimed to study the effect of miR-27 on autophagy in MM via NEDD4/Notch1 axis. RT-qPCR or western blot analysis was used to detect the expression of miR-27, NEDD4, and Notch1 in bone marrow tissues and CD138+ plasma cells of patients and MM cells. After gain- and loss-of-function assays in MM cells, proliferation and invasion were assessed by clone formation and Transwell assays. Meanwhile, expression of autophagy-related proteins was measured by western blot analysis, followed by evaluation of autophagosomes and autophagic flow. The targeting relationship was evaluated by luciferase reporter assay, whereas the binding of NEDD4 to Notch1 protein was analyzed by co-immunoprecipitation. The ubiquitination level of Notch1 protein was detected. A nude mouse tumor model was established to determine the role of miR-27 in MM in vivo. miR-27 and Notch1 upregulation and NEDD4 downregulation were observed in bone marrow tissues and CD138+ plasma cells of patients and MM cells. miR-27 negatively targeted NEDD4, while NEDD4 could specifically bind to Notch1 protein to increase Notch1 ubiquitin degradation in MM cells. miR-27 or Notch1 overexpression or NEDD4 silencing diminished autophagy but enhanced proliferation and invasion of MM cells. miR-27 upregulation promoted the formation of subcutaneous tumor in nude mice. Collectively, miR-27 elevated Notch1 expression by targeting NEDD4 and promoted the development of MM by inhibiting cell autophagy, which provides a new idea and basis for MM treatment.

Synthesis of sophocarpine triflorohydrazone and its proliferation inhibition and apoptosis induction activity in myeloma cells through Notch3-p53 signaling activation.

Multiple myeloma is indicated by the presence of excessive monoclonal plasma cells in bone marrow, which result in the formation of osteolytic lesions. The present study investigated SCA as anti-proliferative agent for myeloma cells and explored the mechanism associated. Effect of SCA on viabilities of KRASA12 and AMO-1 cells was evaluated by MTT assay and apoptotic ratio using flow cytometry. Protein expression was investigated by western blotting and expression of genes related to Notch3-p53 signaling axis using RT-PCR assay. Increase in SCA concentration caused a significant (P < .01) reduction in KRASA12 and AMO-1 cell viability. The KRASA12 and AMO-1 cell viabilities were reduced to 29% and 21%, respectively on treatment with 21 μM doses of SCA. SCA treatment of KRASA12 and AMO-1 cells significantly (P < .05) increased apoptosis compared with untreated cells. The Bcl-2 (26 kDa) protein expression was reduced whereas the Bax (21 kDa) and cleaved caspase-3 levels elevated in SCA treated KRASA12 and AMO-1 cells. Treatment with SCA significantly promoted Hes1, p53 (53 kDa) and Hey1 mRNA expression in KRASA12 and AMO-1 cells. Treatment of KRASA12 and AMO-1 cells with SCA led to a marked reduction in Notch3 protein expression. SCA inhibits KRASA12 and AMO-1 myeloma cell proliferation by promoting pro-apoptotic proteins. Moreover, SCA treatment suppressed Hes1 and Hey1 mRNA expression and targeted Notch3 expression. Therefore, SCA may be studied further for development of treatment for myeloma.

The DLC-1 tumor suppressor is involved in regulating immunomodulation of human mesenchymal stromal /stem cells through interacting with the Notch1 protein

BackgroundImmunomodulatory activities of human mesenchymal stromal /stem cells (hMSCs) has been widely recognized as the most critical function of hMSCs for exerting its therapeutic effects. However, the detailed mechanisms responsible for regulating the immunomodulation of hMSCs still remain largely unknown. Previous studies revealed that the Notch1 protein exerted a pro-immunomodulatory function probably through interacting with the protein(s) subjective to proteasome-mediated protein degradation. The DLC-1 protein represents a well characterized tumor suppressor subjective to proteasome-mediated degradation. However, the detailed signaling pathway of Notch1 and the involvement of DLC-1 in regulating the immunomodulation of hMSCs have not been studied before.MethodsThe transfection with cDNA or siRNA into hMSCs assisted by co-culture of hMSCs with peripheral blood mononuclear cells and small molecule inhibitors of signaling proteins, followed by immunoprecipitation, Western blotting, RT-PCR, and flowcytometry, were employed to characterize the Notch1 signaling, to identify DLC-1 as a candidate proteasome-targeted protein, and to characterize DLC-1 signaling pathway and its interaction with the Notch1 signaling, in the regulation of immunomodulation of hMSCs, specifically, the inhibition of pro-inflammatory CD4+-Th1 lymphocytes, and the release of immunomodulatory molecule IDO1.Statistical analysisOne-way ANOVA was utilized as a statistical tool to analyze the data presented as means ± SEM of at least three separate experiments.ResultsThe present study revealed that the Notch1-Hey1 axis, but not the Notch1-Hes1 axis, was likely responsible for mediating the pro-immunomodulatory function of the Notch1 signaling. The DLC-1 protein was found subjective to proteasome-mediated protein degradation mediated by the DDB1 and FBXW5 E3 ligases and served as an inhibitor of the immunomodulation of hMSCs through inhibiting Rock1, but not Rock2, downstream the DLC-1 signaling. The Notch1 signaling in the Notch1-Hey1 pathway and the DLC-1 signaling in the DLC-1-Rock1-FBXW5 pathway exhibited a mutual exclusion interaction in the regulation of immunomodulation of hMSCs.ConclusionsThe present study uncovers a novel function of DLC-1 tumor suppressor in regulating the immunomodulation of hMSCs. It also proposes a novel mutual exclusion mechanism between the DLC-1 signaling and the Notch1 signaling that is possibly responsible for fine-tuning the immunomodulation of hMSCs with different clinical implications in hMSCs therapy.

Contribution of Autophagy-Notch1-Mediated NLRP3 Inflammasome Activation to Chronic Inflammation and Fibrosis in Keloid Fibroblasts.

Keloid is a representative chronic fibroproliferative condition that occurs after tissue injury. Emerging evidence showed that activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is involved in the pro-inflammatory response in injured tissues. However, the role of NLRP3 inflammasome in keloid progression remains unclear. Notch signaling, which activates NLRP3 inflammasome, is known to contribute to scar formation in keloid, but the cause of enhanced Notch signaling in keloid is not clear. We sought to investigate whether autophagy regulates Notch1 signaling in keloid fibroblasts and determine whether Notch1 signaling might regulate NLRP3 inflammasomes and myofibroblast differentiation. An in vitro model of keloid was established by culturing primary keloid fibroblasts from patients. Expression levels of Notch1, NLRP3 inflammasome proteins, pro-inflammatory cytokines, and myofibroblast markers in keloid fibroblasts were examined and compared with those in normal fibroblasts. Autophagy known to mediate Notch1 degradation was also monitored in fibroblasts. Small interfering RNA (siRNA) targeting Notch1 was used to transfect keloid fibroblasts to further examine the role of Notch signaling in NLRP3 inflammasome activation. Expression levels of Notch1 and NLRP3 inflammasome in keloid fibroblasts increased compared to those in normal fibroblasts. Such increases were accompanied by increased LC3 levels and reduced autophagic flux. Notch1 silencing in keloid fibroblasts by siRNA transfection significantly suppressed increased levels of overall NLRP3 inflammasome complex proteins, NF-kB, and α-smooth muscle actin. Autophagy induction by rapamycin treatment in keloid fibroblasts effectively suppressed expression levels of Notch1 and NLRP3 inflammasome proteins. Decreased autophagy activity in keloid can result in Notch1-mediated myofibroblast activation and NLRP3 inflammasome signaling activation which is critical for chronic inflammation. Collectively, these results identify Notch1 as a novel activator of NLRP3 inflammasome signaling leading to chronic tissue damage and myofibroblast differentiation in keloid progression.

The prognostic implications of Notch1, Hes1, Ascl1, and DLL3 protein expression in SCLC patients receiving platinum-based chemotherapy.

ObjectivesThe aim was to analyse the tumor expression of Notch1, Hes1, Ascl1, and DLL3 in Small-Cell Lung Cancer (SCLC) and each such biomarker’s potential association with clinical characteristics and prognosis after platinum-doublet chemotherapy (PDCT).Material and methodsThe protein expression of the biomarkers was evaluated using immunohistochemistry. Patients were categorized according to their sensitivity to first line PDCT: with a Progression-free survival (PFS) ≥ 3 months after completion of treatment considered “sensitive” and < 3 months after completion of treatment considered “refractory”. PFS and overall survival were computed using Kaplan-Meier curves with 95% confidence interval.Results and conclusionThe study included 46 patients, with 21 and 25 of the patients having “sensitive” and “refractory” disease, respectively. The majority of patients had a high DLL3 expression (n = 38), while a minority had Notch 1-high expression (n = 10). The chi-square test showed that there was a statistically significant negative association between Notch1 and Ascl1 expression (p = 0.013). The overall survival for patients with Notch1- high vs. low expression was 8.1 vs. 12.4 months, respectively (p = 0.036). Notch1 expression was an independent prognostic factor in the multivariate analysis (p = 0.02). No other biomarker showed any prognostic impact in this highly selected SCLC cohort. DLL3 is highly expressed in the majority of advanced staged SCLC cases, as expected. In the same patient population, Notch1 expression might have a potential prognostic implication, by driving a non-neuroendocrine differentiation process. Given the small number of cases with Notch1 high expression, the results of this study needs to be confirmed on a larger cohort.

Identifying specific Notch1 target proteins in lung carcinoma cells.

The Notch signaling pathway has different roles in many human neoplasms, being either tumor-promoting or anti-proliferative. In addition, Notch signaling in carcinogenesis can be tissue dependent. The aim of the current study is to elucidate the relation between Notch1 protein expression in lung cancer cells and the following Notch related proteins: Hes1, c-Myc, Jagged1 and Jagged2. Notch1 and its related proteins were detected in human lung cancer cell lines and in 54 surgically resected different lung carcinoma tissues. Then, we used small interfering RNA (siRNA) technology, to down-regulate the expression of Notch1 in H69AR and SBC3 small cell lung carcinoma (SCLC) cells. Also, we transfected venus Notch1 intracellular domain (v.NICD) plasmid into human SCLC lines; H69. The expression of Hes1, c-Myc and Jagged2 is affected by Notch1 in SCLC. There is a strong association between the expression of Notch1 protein and the expression of Hes1, c-Myc and Jagged2 proteins, which could aid in better understanding tumorigenesis in SCLC.

Endothelial Notch1 Regulates High-dose Radiation Therapy (HDRT)-induced Endothelial-to-mesenchymal Transition (EbdMT)

There is increasing evidence that high-dose radiation therapy (HDRT) induces endothelial-to-mesenchymal-transition (EdnMT) which promotes abnormal tumor vascularity and radioresistance. We have previously shown that HDRT induces Notch1 signaling in tumor endothelial cells. Here, we investigate if endothelial Notch1 regulates HDRT induced EdnMT. HUVEC monolayers, in vitro, were also treated with 0, 2, 8, and 12Gy and immunoblot was performed for Notch1, Jagged1, VE-Cadherin. HUVEC monolayers were also treated with a soluble Notch inhibitor, Notch1 decoy, to assess the effect of Notch inhibition on EdnMT. Human neuroblastoma cells NGP were engineered to express Fc(control) and Notch1 decoy. These cells were intrarenally injected to develop xenograft tumors. Tumors were treated with 0 Gy and 12 Gy single fraction doses of HDRT. Mice were sacked at 72 hr after HDRT. Tumor sections were subjected to double immunofluorescence staining for aSMA (mesenchymal marker) and endomucin (endothelial marker). We also assessed the effect of combining HDRT with Notch inhibition on macrophage infiltration into the tumor microenvironment by performing immunofluorescence staining for F480 (a marker for tumor associated macrophage), INOS (marker for M1 macrophage) and MRC1 (a marker for M2 macrophage). In HUVEC monolayers, HDRT doses of 8 Gy and 12 Gy profoundly increased Notch1 and Jagged1 protein levels after 72 hr. The increased level of Notch1 and Jagged1 was associated with the reduction of endothelial marker VE-Cadherin level. Notch1 decoy treatment inhibited reduction of VE-cadherin indicating Notch inhibition blocked EdnMT. In vivo in NGP xenograft mice model, increased colocalization of endomucin and αSMA was observed at 72 hr 12 Gy post-irradiation, when compared to nonirradiated tumors, indicating transition of ECs. Interestingly, Notch inhibition reduced colocalizing αSMA (+) endomucin (+) staining in Notch1decoy+12 Gy tumors. Quantification showed that while ∼4% and ∼3% of endomucin (+) vessels expressed αSMA, following Fc+0 Gy and Notch1decoy + 0 Gy treatments, respectively, ∼30% of the surviving vessels were αSMA (+) after Fc+12 Gy. Notch inhibition reduced αSMA staining in surviving vessels as ∼4% of the vessels were αSMA (+) in N11-24-decoy+12 Gy tumors. Increased F480 and MRC1 staining was observed in tumor treated with 12 Gy, compared to untreated tumors, while no change in INOS staining was noted. Notch1 decoy treatment significantly decreased F480 and MRC1 suggesting Notch blockade decreased HDRT induced M2 macrophage infiltration. Our data shows that Notch blockade inhibits HDRT-induced EdnMT. Our data further shows that HDRT increased tumor-promoting immune suppressive M2 macrophage infiltration. Combing HDRT with Notch blockade inhibits M2 macrophage infiltration into the tumor microenvironment and can be a potential therapeutic approach to minimize immunosuppressive immune response.

Effect of Notch pathway on lipid accumulation induced by mono-2-ethylhexyl phthalate on 3T3-L1 cells

BackgroundMono-2-ethylhexyl phthalate (MEHP) is a major metabolite of di (2-ethylhexyl) phthalate (DEHP). Our previous researches have shown that MEHP can induce lipid accumulation in preadipocytes, while, the underlying mechanism is unclear. The present study was undertaken to clarify the effect of Notch pathway on lipid accumulation induced by MEHP. Methods3T3-L1 preadipocytes were exposed to MEHP (0, 10, 50, 250 µM and 0.1%DMSO) for the whole differentiation phase. Then the level of TG and cell cycle were detected. RT-PCR was used to detect the mRNA expression and Western blot was used to detect the expression of protein by Notch pathway genes and lipid metabolic related genes. ResultsIn this study, the level of TG in the 250 µM and 250 µM MEHP groups was significantly higher than that in the control, DMSO and 10 µM MEHP groups (P < 0.05). The relative mRNA level of Notch-1, Notch-3, Notch-4, Jagged-2 and Dll-4 in 250 µM group was higher than other groups (P < 0.05). The expression of Notch signal pathway proteins increased in MEHP treated groups, and the expression of Notch-2, Jagged-1, Jagged-2, Dll-1 and Dll-4 in 250 µM group were significantly higher than control group (P < 0.05). The expression of lipid metabolic related gene mRNA and protein increased in MEHP treated groups, and 250 µM MEHP group was higher than other groups (P < 0.05). The intracellular TG content was significantly correlated with the expression levels of Notch-1 and Jagged-2 mRNA (P < 0.05). ConclusionIn this study, we have found that MEHP exposure could increase the TG content in 3T3-L1 cells. The expression of Notch pathway mRNA and proteins were disturbed by the MEHP. Notch-1 and its ligand Jagged-2 play a critical role in the abnormal lipid metabolism in 3T3-L1 cells caused by MEHP.

Roads to the strategic targeting of ovarian cancer treatment.

Although ovarian cancer mortality rates have slightly declined in the last 40 years, ovarian cancer continues to be the eighth cause of cancer death in women. Ovarian cancer is characterized by its high response to treatments but also by its high rate of recurrence. Although treatments are limited to cytoreductive surgery and platinum-based chemotherapy, other therapies using antiangiogenic agents and poly (ADP-ribose) polymerase inhibitors are being tested. Nevertheless, these therapeutic strategies have had poor results and new potential targets and approaches are thus needed. The present review focuses on the recent evidence on antiangiogenic strategies in ovarian cancer cells and on the mechanisms governed by Notch and β-catenin proteins. It also describes the concept of 'vascular normalization' by using the platelet-derived growth factor, PDGFB, molecule as a tool to regulate ovarian tumor angiogenesis and thus improve ovarian tumor treatment. It has been reported that alterations in the Notch system components and changes in the canonical Wnt/β-catenin signaling, the other pathway of our interest, are relevant to molecular events that contribute to ovarian cancer development. Thus, in this review, we consider these aspects of the ovarian tumor biology as potential new therapeutic strategies for the treatment of this disease.

Targeting Notch Trafficking and Processing in Cancers

The Notch family comprises a group of four ligand-dependent receptors that control evolutionarily conserved developmental and homeostatic processes and transmit signals to the microenvironment. NOTCH undergoes remodeling, maturation, and trafficking in a series of post-translational events, including glycosylation, ubiquitination, and endocytosis. The regulatory modifications occurring in the endoplasmic reticulum/Golgi precede the intramembrane γ-secretase proteolysis and the transfer of active NOTCH to the nucleus. Hence, NOTCH proteins coexist in different subcellular compartments and undergo continuous relocation. Various factors, including ion concentration, enzymatic activity, and co-regulatory elements control Notch trafficking. Interfering with these regulatory mechanisms represents an innovative therapeutic way to bar oncogenic Notch signaling. In this review, we briefly summarize the role of Notch signaling in cancer and describe the protein modifications required for NOTCH to relocate across different subcellular compartments. We focus on the functional relationship between these modifications and the corresponding therapeutic options, and our findings could support the development of trafficking modulators as a potential alternative to the well-known γ-secretase inhibitors.

Notch3 and DeltaB maintain Müller glia quiescence and act as negative regulators of regeneration in the light-damaged zebrafish retina.

Damage to the zebrafish retina stimulates resident Müller glia to reprogram, reenter the cell cycle, divide asymmetrically, and produce neuronal progenitor cells that amplify and differentiate into the lost neurons. The transition from quiescent to proliferative Müller glia involves both positive and negative regulators. We previously demonstrated that the Notch signaling pathway represses retinal regeneration by maintaining Müller glia quiescence in zebrafish. Here we examine which Notch receptor is necessary to maintain quiescence. Quantitative RT-PCR and RNA-Seq analyses reveal that notch3 is expressed in the undamaged retina and is downregulated in response to light damage. Additionally, Notch3 protein is expressed in quiescent Müller glia of the undamaged retina, is downregulated as Müller glia proliferate, and is reestablished in the Müller glia. Knockdown of Notch3 is sufficient to induce Müller glia proliferation in undamaged retinas and enhances proliferation during light damage. Alternatively, knockdown of Notch1a, Notch1b, or Notch2 decreases the number of proliferating cells during light damage, suggesting that Notch signaling is also required for proliferation during retinal regeneration. We also knockdown the zebrafish Delta and Delta-like proteins, ligands for the Notch receptors, and find that the deltaB morphant possesses an increased number of proliferating cells in the light-damaged retina. As with Notch3, knockdown of DeltaB is sufficient to induce Müller glia proliferation in the absence of light damage. Taken together, the negative regulation of Müller glia proliferation in zebrafish retinal regeneration is mediated by Notch3 and DeltaB.

Integrin Beta 1 Promotes Glioma Cell Proliferation by Negatively Regulating the Notch Pathway.

In this study, genes associated with the Notch signaling pathway in gliomas were analyzed using bioinformatics and in vitro experiments. The dataset GSE22772 was downloaded from the Gene-Cloud of Biotechnology Information database. Differentially expressed genes (DEGs) between short hairpin RNA (shRNA) intervening glioma cells and control cells were screened using the unpaired t test. Functional enrichment analysis was performed, and coexpression network was analyzed to identify the most important genes associated with the Notch signaling pathway. Integrin beta 1 (ITGB1) mRNA and protein levels in clinical glioma tumor samples and tumor adjacent normal tissue samples were analyzed using quantitative real-time PCR and immunohistochemistry, respectively. The relationship between ITGB1 expression and the prognosis of patients with gliomas was analyzed using the Kaplan-Meier curve. ITGB1 interference expression cell line U87 and ITGB1 overexpressing cell line were established using sh-ITGB1 and oe-ITGB1 plasmids, respectively. MTT and colony formation assays were used to detect changes in the proliferation of glioma cells. Moreover, western blotting was used to detect the expression of Notch and Hey1. A total of 7,962 DEGs were screened between shRNA intervening glioma cells and control cells, which were mainly associated with spliceosome, proteoglycans in cancer, focal adhesion, and the Notch signaling pathway. ITGB1 showed the highest expression in the coexpression network. The mRNA and protein expression of ITGB1 in glioma tumor samples was significantly higher than that in tumor adjacent normal tissue samples (p < 0.05). Overall survival time of patients in the ITGB1 low-expression group was significantly longer than that in the ITGB1 high-expression group, indicating that ITGB1 expression negatively correlated with the prognosis. Fluorescence microscopy, qRT-PCR, and western blotting confirmed the transfection efficiency of ITGB1 overexpression and interference expression in U251 and U87 cells. The MTT and colony formation assays indicated that U87 cell proliferation was significantly inhibited after intervention with ITGB1 (p < 0.05), and overexpression of ITGB1 significantly promoted U251 cell proliferation (p < 0.05). In addition, the expression of Notch and Hey1 proteins was significantly decreased after ITGB1 intervention (p < 0.05), and their expression was significantly upregulated after ITGB1 overexpression (p < 0.05). ITGB1 expression in glioma tissues was significantly higher than that in adjacent normal tissues and was negatively correlated with the survival time of patients. Therefore, ITGB1 can significantly promote proliferation of glioma cells via feedback regulation of the Notch signaling pathway.

LncRNA-NEAT1 promotes proliferation of T-ALL cells via miR-146b-5p/NOTCH1 signaling pathway

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is a malignant tumor of the hematopoietic system, which can develop at any age, with the symptoms of weakness, fatigue, enlarged lymph nodes, or weight loss. Nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in the process of T-ALL, but the regulatory mechanism is still not known clearly. MethodsThe expression levels of NEAT1 and miR-146b-5p in T-ALL cells were performed by qRT-PCR and NOTCH1 protein level- wwWwas determined by western blot assay. Dual-luciferase reporter assay was used to detect the interaction between NEAT1 and miR-146b-5p, as well as miR-146b-5p and NOTCH1. The cell proliferation was measured by using MTT assay and colony formation assay. ResultsThe expression levels of NEAT1 were markedly increased, but miR-146b-5p levels were reduced in T-ALL cells. Knockdown of NEAT1 or overexpression of miR-146b-5p decreased NOTCH1 expression, inhibited the proliferation of T-ALL cells. MiR-146b-5p bound both NEAT1 and NOTCH1 3′-UTR directly. Finally, inhibition of miR-146b-5p could abrogate the effects of NEAT1 knockdown on the proliferation of T-ALL cells. ConclusionNEAT1 promotes the proliferation of T-ALL cells by sponging miR-146b-5p to upregulate the expression of NOTCH1. The results of this study provide new insight into the action mechanism of NEAT1 modulating T-ALL progression.

Dual inhibition of Src and PLK1 regulate stemness and induce apoptosis through Notch1-SOX2 signaling in EGFRvIII positive glioma stem cells (GSCs)

Glioma stem cells (GSCs) have been implicated in the promotion of malignant progression. Epidermal growth factor receptor variant (EGFRv) has been associated with glioma “stemness”. However, the molecular mechanism is not clear. In this study, we were committed to investigate the role of EGFRv in GSCs and presented a new therapeutic target in EGFRvIII positive GSCs. The results showed that EGFRvIII could induce the expression of p-Src and PLK1, and both could induce the Notch1-SOX2 signaling pathway to promote self-renewal and tumor progression of GSCs. Mechanistically, both p-Src and PLK1 can induce Notch1, and the intracellular domain of Notch1 (NICD) can directly bind to SOX2, thereby promoting the maintenance of glioma stem cells. Furthermore, Saracatinib (Src inhibition) and BI2536 (PLK1 inhibition) diminished GSC self-renewal in vitro, and combining the two inhibitors increased survival of orthotopic tumor-bearing mice. Taken together, these data indicate that p-Src and PLK1 contribute to cancer stemness in EGFRvIII-positive GSCs by driving Notch1-SOX2 signaling, a finding that has important clinical implications.

HYPOXIA CONDITION MODIFIES THE EXPRESSION OF HIF-1α AND NOTCH-1 PROTEINS IN AMELOBLASTOMA CELL LINES

Ameloblastoma is one of the most prevalent odontogenic tumors, with high rates of recurrence and invasiveness. Oxygen reduction favors neoplastic cell adaptation to be capable of overcoming oxygen deprivation, which can increase invasion. Objective: The aim of this study was to evaluate the expression of the proteins HIF-1α and NOTCH-1, related to the invadopodia formation, which are cell structures present in tumor invasion, comparing the markings in normoxia and hypoxia conditions for 1, 2, 4, and 8 hours. Study Design: The methods used were cell viability assay (MTT), hypoxia assay, and indirect immunofluorescence assay. Results: The MTT method revealed that in hypoxic conditions at 1, 2, 4, and 8 hours, the cells were viable. Regarding the expression of the proteins studied, in immunofluorescence assay, greater immunoexpression in hypoxia compared with normoxia was observed. Conclusion: It is concluded, therefore, that the decrease in the oxygen supply (1) favors a greater expression of HIF-1α and NOTCH-1 proteins in the ameloblastoma cell line, (2) does not modify cell viability, and (3) results in metabolic alteration of the cells. Possibly, the greater expression of the proteins studied under hypoxic conditions may be related to the local invasion of the tumor. Ameloblastoma is one of the most prevalent odontogenic tumors, with high rates of recurrence and invasiveness. Oxygen reduction favors neoplastic cell adaptation to be capable of overcoming oxygen deprivation, which can increase invasion. Objective: The aim of this study was to evaluate the expression of the proteins HIF-1α and NOTCH-1, related to the invadopodia formation, which are cell structures present in tumor invasion, comparing the markings in normoxia and hypoxia conditions for 1, 2, 4, and 8 hours. Study Design: The methods used were cell viability assay (MTT), hypoxia assay, and indirect immunofluorescence assay. Results: The MTT method revealed that in hypoxic conditions at 1, 2, 4, and 8 hours, the cells were viable. Regarding the expression of the proteins studied, in immunofluorescence assay, greater immunoexpression in hypoxia compared with normoxia was observed. Conclusion: It is concluded, therefore, that the decrease in the oxygen supply (1) favors a greater expression of HIF-1α and NOTCH-1 proteins in the ameloblastoma cell line, (2) does not modify cell viability, and (3) results in metabolic alteration of the cells. Possibly, the greater expression of the proteins studied under hypoxic conditions may be related to the local invasion of the tumor.