Prognostic value of CD8+T cells related genes and exhaustion regulation of Notch signaling pathway in hepatocellular carcinoma.

Immunotherapy has emerged as the primary treatment modality for patients with advanced Hepatocellular carcinoma (HCC). However, its clinical efficacy remains limited, benefiting only a subset of patients, while most exhibit immune tolerance and face a grim prognosis. The infiltration of immune cells plays a pivotal role in tumor initiation and progression. In this study, we conducted an analysis of immune cell infiltration patterns in HCC patients and observed a substantial proportion of CD8+T cells. Leveraging the weighted gene co-expression network analysis (WGCNA), we identified 235 genes associated with CD8+T cell and constructed a risk prediction model. In this model, HCC patients were stratified into a high-risk and low-risk group. Patients in the high-risk group exhibited a lower survival rate, predominantly presented with intermediate to advanced stages of cancer, displayed compromised immune function, showed limited responsiveness to immunotherapy, and demonstrated elevated expression levels of the Notch signaling pathway. Further examination of clinical samples demonstrated an upregulation of the Notch1+CD8+T cell exhaustion phenotype accompanied by impaired cytotoxicity and cytokine secretion functions that worsened with increasing Notch activation levels. Our study not only presents a prognostic model but also highlights the crucial involvement of the Notch pathway in CD8+T cell exhaustion-a potential target for future immunotherapeutic interventions.

Regulation of Notch signaling by non-muscle myosin II Zipper in Drosophila.

The Notch pathway is an evolutionarily conserved signaling system that is intricately regulated at multiple levels and it influences different aspects of development. In an effort to identify novel components involved in Notch signaling and its regulation, we carried out protein interaction screens which identified non-muscle myosin II Zipper (Zip) as an interacting partner of Notch. Physical interaction between Notch and Zip was further validated by co-immunoprecipitation studies. Immunocytochemical analyses revealed that Notch and Zip co-localize within same cytoplasmic compartment. Different alleles of zip also showed strong genetic interactions with Notch pathway components. Downregulation of Zip resulted in wing phenotypes that were reminiscent of Notch loss-of-function phenotypes and a perturbed expression of Notch downstream targets, Cut and Deadpan. Further, synergistic interaction between Notch and Zip resulted in highly ectopic expression of these Notch targets. Activated Notch-induced tumorous phenotype of larval tissues was enhanced by over-expression of Zip. Notch-Zip synergy resulted in the activation of JNK pathway that consequently lead to MMP activation and proliferation. Taken together, our results suggest that Zip may play an important role in regulation of Notch signaling.

Upregulation of Notch Signaling and Cell-Differentiation Inhibitory Transcription Factors in Stable Chronic Obstructive Pulmonary Disease Patients.

Notch signaling is involved in the prevention of cell differentiation and cell fate in various organs, including the lungs. We aimed to determine the transcriptomic and protein expression of Notch receptors, their ligands, and related transcription factors in stable COPD. The expression and localization of Notch receptors, their ligands, and related transcription factors were measured in bronchial biopsies of individuals with stable mild/moderate (MCOPD) (n = 18) or severe/very severe (SCOPD) (n = 16) COPD, control smokers (CSs) (n = 13), and control nonsmokers (CNSs) (n = 11), and in the lung parenchyma of those with MCOPD (n = 13), CSs (n = 10), and CNSs (n = 10) using immunohistochemistry, ELISA tests, and transcriptome analyses. In the bronchial biopsies, Notch4 and HES7 significantly increased in the lamina propria of those with SCOPD compared to those with MCOPD, CSs, and CNSs. In the peripheral lung bronchiolar epithelium, Notch1 significantly increased in those with MCOPD and CSs compared to CNSs. ELISA tests of lung parenchyma homogenates showed significantly increased Notch2 in those with MCOPD compared to CSs and CNSs. Transcriptomic data in lung parenchyma showed increased DLL4 and HES1 mRNA levels in those with MCOPD and CSs compared to CNSs. These data show the increased expression of the Notch pathway in the lungs of those with stable COPD. These alterations may play a role in impairing the regenerative-reparative responses of diseased bronchioles and lung parenchyma.

Evolution and Function of the Notch Signaling Pathway: An Invertebrate Perspective.

The highly conserved Notch signaling pathway affects embryonic development, neurogenesis, homeostasis, tissue repair, immunity, and numerous other essential processes. Although previous studies have demonstrated the location and function of the core components of Notch signaling in various animal phyla, a more comprehensive summary of the Notch core components in lower organisms is still required. In this review, we objectively summarize the molecular features of the Notch signaling pathway constituents, their current expression profiles, and their functions in invertebrates, with emphasis on their effects on neurogenesis and regeneration. We also analyze the evolution and other facets of Notch signaling and hope that the contents of this review will be useful to interested researchers.

Force-free activation of Notch with DNA origami.

The Notch signaling pathway is a highly conserved, fundamental process to embryogenesis and neurogenesis. While force-induced conformational change is known to activate Notch receptors, Smyrlaki et al. recently used DNA origami to reveal an additional, force-independent mode of Notch activation via soluble presentation of spatially controlled ligand nanopatterns.

Unraveling paralog-specific Notch signaling through thermodynamics of ternary complex formation and transcriptional activation of chimeric receptors.

Notch signaling in humans is mediated by four paralogous receptors that share conserved architectures and possess overlapping, yet non-redundant functions. The receptors share a canonical activation pathway wherein upon extracellular ligand binding, the Notch intracellular domain (NICD) is cleaved from the membrane and translocates to the nucleus where its N-terminal RBP-j-associated molecule (RAM) region and ankyrin repeat (ANK) domain bind transcription factor CSL and recruit co-activator Mastermind-like-1 (MAML1) to activate transcription. However, different paralogs can lead to distinct outcomes. To better understand paralog-specific differences in Notch signaling, we performed a thermodynamic analysis of the Notch transcriptional activation complexes for all four Notch paralogs using isothermal titration calorimetry. Using chimeric constructs, we find that the RAM region is the primary determinant of stability of binary RAMANK:CSL complexes, and that the ANK regions are largely the determinants of MAML1 binding to pre-formed RAMANK:CSL complexes. Free energies of these binding reactions (ΔGRA and ΔGMAML) vary among the four Notch paralogs, although variations for Notch2, 3, and 4 offset in the free energy of the ternary complex (ΔGTC, where ΔGTC = ΔGRA + ΔGMAML). To probe how these affinity differences affect Notch signaling, we performed transcriptional activation assays with the paralogous and chimeric NICDs, and analyzed the results with an independent multiplicative model that quantifies contributions of the paralogous RAM, ANK, and C-terminal regions (CTR) to activation. This analysis shows that transcription activation correlates with ΔGTC, but that activation is further modified by CTR identity in a paralog-specific way.

Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL.

Loss of arterial smooth muscle cells (SMCs) and abnormal accumulation of the extracellular domain of the NOTCH3 receptor (Notch3ECD) are the 2 core features of CADASIL, a common cerebral small vessel disease caused by highly stereotyped dominant mutations in NOTCH3. Yet the relationship between NOTCH3 receptor activity, Notch3ECD accumulation, and arterial SMC loss has remained elusive, hampering the development of disease-modifying therapies. Using dedicated histopathological and multiscale imaging modalities, we could detect and quantify previously undetectable CADASIL-driven arterial SMC loss in the CNS of mice expressing the archetypal Arg169Cys mutation. We found that arterial pathology was more severe and Notch3ECD accumulation greater in transgenic mice overexpressing the mutation on a wild-type Notch3 background (TgNotch3R169C) than in knockin Notch3R170C/R170C mice expressing this mutation without a wild-type Notch3 copy. Notably, expression of Notch3-regulated genes was essentially unchanged in TgNotch3R169C arteries. We further showed that wild-type Notch3ECD coaggregated with mutant Notch3ECD and that elimination of 1 copy of wild-type Notch3 in TgNotch3R169C was sufficient to attenuate Notch3ECD accumulation and arterial pathology. These findings suggest that Notch3ECD accumulation, involving mutant and wild-type NOTCH3, is a major driver of arterial SMC loss in CADASIL, paving the way for NOTCH3-lowering therapeutic strategies.

Identifications of three novel alleles of Serrate in Drosophila.

The Notch signaling pathway, an evolutionarily highly conserved pathway, participates in various essential physiological processes in organisms. Activation of Notch signaling in the canonical manner requires the combination of ligand and receptor. There are two ligands of Notch in Drosophila: Delta (Dl) and Serrate (Ser). A mutation mf157 is identified for causing nicks of fly wings in genetic analysis from a mutant library (unpublished) that was established previously. Immunofluorescent staining illustrates that mf157 represses the expression of Cut and Wingless (Wg), the targets of Notch signaling. MARCM cloning analysis reveals that mf157 functions at the same level or the upstream of ligands of Notch in signaling sending cells. Sequencing demonstrates that mf157 is a novel allele of the Ser gene. Subsequently, mf553 and mf167 are also identified as new alleles of Ser from our library. Furthermore, the complementary assays and the examination of transcripts confirm the sequencing results. Besides, the repressed phenotypes of Notch signaling were reverted by transposon excision experiments of mf157. In conclusion, we identify three fresh alleles of Ser. Our works supply additional genetic resources for further study of functions of Ser and Notch signaling regulation.

Notch signaling and Bsh homeodomain activity are integrated to diversify Drosophila lamina neuron types.

Notch signaling is an evolutionarily conserved pathway for specifying binary neuronal fates, yet how it specifies different fates in different contexts remains elusive. In our accompanying paper, using the Drosophila lamina neuron types (L1-L5) as a model, we show that the primary homeodomain transcription factor (HDTF) Bsh activates secondary HDTFs Ap (L4) and Pdm3 (L5) and specifies L4/L5 neuronal fates. Here we test the hypothesis that Notch signaling enables Bsh to differentially specify L4 and L5 fates. We show asymmetric Notch signaling between newborn L4 and L5 neurons, but they are not siblings; rather, Notch signaling in L4 is due to Delta expression in adjacent L1 neurons. While Notch signaling and Bsh expression are mutually independent, Notch is necessary and sufficient for Bsh to specify L4 fate over L5. The NotchON L4, compared to NotchOFF L5, has a distinct open chromatin landscape which allows Bsh to bind distinct genomic loci, leading to L4-specific identity gene transcription. We propose a novel model in which Notch signaling is integrated with the primary HDTF activity to diversify neuron types by directly or indirectly generating a distinct open chromatin landscape that constrains the pool of genes that a primary HDTF can activate.

A novel search engine for proteins involved in Notch crosstalk signaling pathways.

To regulate biological activity in humans, the Notch signaling pathway (NSP) plays an essential role in a wide array of cellular development and differentiation process. In recent years, many studies have reported that aberrant activation of Notch is associated with the tumor process; but no appropriate database exists to fill this significant gap. To address this, we created a pioneering database NCSp, which is open access and comprises intercommunicating pathways and related protein mutations. This allows scientists to understand better the cause of single amino acid mutations in proteins. Therefore, NCSp provides information on the predicted functional effect of human protein mutations, which aids in understanding the importance of mutations linked to the Notch crosstalk signaling pathways in cancerous and non-cancerous systems. This database might be helpful for therapeutic mutation analysis, molecular biology, and structural biology researchers. The NCSp database can be accessed through https://bioserver3.physics.iisc.ac.in/cgi-bin/nccspd/.

Notch intracellular domains form transcriptionally active heterodimeric complexes on sequence-paired sites.

Notch signaling is universally conserved in metazoans where it is important for a wide variety of both normal and abnormal physiology. All four mammalian Notch receptors are activated by a conserved mechanism that releases Notch intracellular domains (NICDs) from the plasma membrane to translocate to the nucleus. Once there, NICDs interact through highly conserved ankyrin domains to form head-to-head homodimers on Notch sensitive promoters and stimulate transcription. Due to the highly conserved nature of these Notch ankyrin domains in all four mammalian Notch proteins, we hypothesized that NICDs may also engage in heterodimerization. Our results reveal the presence of two NICD dimerization states that can both engage in homo and heterodimerization. Using a Co-IP approach, we show that all NICD's can form non-transcriptionally active dimers and that the N4ICD appears to perform this function better than the other NICDs. Using a combination of ChIP analysis and transcriptional reporter assays, we also demonstrate the formation of transcriptionally active heterodimers that form on DNA. In particular, we demonstrate heterodimerization between the N2ICD and N4ICD and show that this heterodimer pair appears to exhibit differential activity on various Notch sensitive promoters. These results illustrate a new diversification of Notch signaling mechanisms which will help us better understand basic Notch function.

NOTCH Signaling Pathway: Occurrence, Mechanism, and NOTCH-Directed Therapy for the Management of Cancer.

It is now well understood that many signaling pathways are vital in carrying out and controlling essential pro-survival and pro-growth cellular functions. The NOTCH signaling pathway, a highly conserved evolutionary signaling pathway, has been thoroughly studied since the discovery of NOTCH phenotypes about 100 years ago in Drosophila melanogaster. Abnormal NOTCH signaling has been linked to the pathophysiology of several diseases, notably cancer. In tumorigenesis, NOTCH plays the role of a "double-edged sword," that is, it may act as an oncogene or as a tumor suppressor gene depending on the nature of the context. However, its involvement in several cancers and inhibition of the same provides targeted therapy for the management of cancer. The use of gamma (γ)-secretase inhibitors and monoclonal antibodies for cancer treatment involved NOTCH receptors inhibition, leading to the possibility of a targeted approach for cancer treatment. Likewise, several natural compounds, including curcumin, resveratrol, diallyl sulfide, and genistein, also play a dynamic role in the management of cancer by inhibition of NOTCH receptors. This review outlines the functions and structure of NOTCH receptors and their associated ligands with the mechanism of the signaling pathway. In addition, it also emphasizes the role of NOTCH-targeted nanomedicine in various cancer treatment strategies.

Treatment with Cerebrolysin Prolongs Lifespan in a Mouse Model of Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare familial neurological disorder caused by mutations in the NOTCH3 gene and characterized by migraine attacks, depressive episodes, lacunar strokes, dementia, and premature death. Since there is no therapy for CADASIL the authors investigate whether the multi-modal neuropeptide drug Cerebrolysin may improve outcome in a murine CADASIL model. Twelve-month-old NOTCH3R169C mutant mice (n=176) are treated for nine weeks with Cerebrolysin or Vehicle and histopathological and functional outcomes are evaluated within the subsequent ten months. Cerebrolysin treatment improves spatial memory and overall health, reduces epigenetic aging, and prolongs lifespan, however, CADASIL-specific white matter vacuolization is not affected. On the molecular level Cerebrolysin treatment increases expression of Calcitonin Gene-Related Peptide (CGRP) and Silent Information Regulator Two (Sir2)-like protein 6 (SIRT6), decreases expression of Insulin-like Growth Factor 1 (IGF-1), and normalizes the expression of neurovascular laminin. In summary, Cerebrolysin fosters longevity and healthy aging without specifically affecting CADASIL pathology. Hence, Cerebrolysin may serve a therapeutic option for CADASIL and other disorders characterized by accelerated aging.

Epigenetically dysregulated NOTCH-Delta-HES signaling cascade can serve as a subtype classifier for acute lymphoblastic leukemia.

The NOTCH-Delta-HES signaling cascade is regarded as a double-edged sword owing to its dual tumor-suppressor and oncogenic roles, in different cellular environments. In the T-cells, it supports leukemogenesis by promoting differentiation while in B-cells, it controls leukemogenesis by inhibiting early differentiation/inducing growth arrest in the lead to apoptosis. The present study was undertaken to assess if this bi-faceted behavior of NOTCH family can be exploited as a diagnostic biomarker or subtype classifier of acute lymphoblastic leukemia (ALL). In this pursuit, expression of seven NOTCH cascade genes was analyzed in bone marrow (BM) biopsy and blood plasma (BP) of pediatric ALL patients using quantitative PCR (qPCR). Further, promoter DNA methylation status of the differentially expressed genes (DEGs) was assessed by methylation-specific qMSP and validated through bisulphite amplicon sequencing. Whereas hypermethylation of JAG1, DLL1, and HES-2, HES-4, and HES-5 was observed in all patients, NOTCH3 was found hypermethylated specifically in Pre-B ALL cases while DLL4 in Pre-T ALL cases. Aberrant DNA methylation strongly correlated with downregulated gene expression, which restored at complete remission stage as observed in "follow-up/post-treatment" subjects. The subtype-specific ROC curve analysis and Kaplan-Meier survival analysis predicted a clinically applicable diagnostic and prognostic potential of the panel. Moreover, the logistic regression model (Pre-B vs Pre-T ALL) was found to be the best-fitted model (McFadden's R2 = 0.28, F1 measure = 0.99). Whether analyzed in BM-aspirates or blood plasma, the NOTCH epigenetic signatures displayed comparable results (p < 0.001), advocating the potential of NOTCH-Delta-HES cascade, as a subtype classifier, in minimally invasive diagnosis of ALL.

PLS3 promotes papillary thyroid carcinoma progression by activating the Notch signaling pathway.

Thyroid cancer is the most common endocrine malignancy worldwide. Although significant progress has been made in understanding the genetic and molecular alterations that drive thyroid cancer, the mechanisms underlying thyroid tumor progression remain unclear. In this study, we explored the involvement of Plastin-3 (PLS3) in the progression of papillary thyroid cancer and elucidated the underlying molecular mechanisms. We first analyzed clinical samples from papillary thyroid cancer patients and found that PLS3 expression was significantly upregulated in tumor tissues compared to adjacent normal tissues. Moreover, high PLS3 expression was associated with advanced tumor stage and poor prognosis. Further in vitro and in vivo experiments showed that PLS3 could promote the proliferation, migration, and invasive behavior of papillary thyroid cancer cells, while PLS3 knockdown suppressed these processes. Mechanistically, we found that PLS3 promoted papillary thyroid cancer progression by activating the Notch signaling pathway. Specifically, PLS3 upregulated the expression of Notch receptors (Notch1) and downstream target gene (Hes1) in papillary thyroid cancer cells. In summary, our findings collectively indicate that PLS3 plays a pivotal role in driving the progression of papillary thyroid cancer and holds promise as a viable therapeutic target for the treatment of this disease.

Modifiable vascular risk factors contribute to stroke in 1080 NOTCH3 R544C carriers in Taiwan Biobank.

BACKGROUND AND AIM: Previous studies have suggested cardiovascular risk factors increase the risk of not only common sporadic stroke but also of stroke in patients with monogenic stroke disorders including CADASIL. We investigated the effects of the NOTCH3 Arg544Cys (R544C) variant and associated vascular risk factors on stroke in the Taiwanese population. METHODS: This study was conducted using data from the Taiwan Biobank, consisting of at least 130,000 Han Chinese participants. The genotype was derived from customized genome-wide arrays for 650,000 to 750,000 single-nucleotide polymorphisms (SNPs). Individuals with NOTCH3 R544C were subsequently matched with noncarriers based on the propensity score at a 1:10 ratio by demographic and cardiovascular risk factors. The odds ratio (OR) for stroke or other phenotypes in NOTCH3 R544C carriers and matched noncarriers was then calculated. Univariate and multivariate regression analyses were performed on cardiovascular risk factors in NOTCH3 R544C carriers with and without stroke. The polygenic risk score (PRS) model, adopted from the UK Biobank, was then applied to evaluate the role of NOTCH3 R544C in stroke. RESULTS: From the 114,282 participants with both genotype and questionnaire results, 1080 (0.95%) harbored the pathogenic NOTCH3 R544C variant. When compared to the matched controls (n = 10,800), the carriers presented with a history of stroke (OR: 2.52, 95% confidence interval (CI) (1.45, 4.37)), dementia (OR: 30.1, 95% CI (3.13, 289.43)), and sibling history of stroke (OR: 2.48, 95% CI (1.85, 3.34)) phenotypes. The risk of stroke increased with every 10-year increase in age (p = 0.006, Cochran-Mantel-Haenszel test). Among NOTCH3 R544C carriers, 16 (1.3%) of the 1080 carriers with a stroke history were older, male, and more likely to have hypertension, diabetes, dyslipidemia, and a family history of stroke. In the stepwise multivariate analysis, hypertension (OR: 11.28, 95% CI (3.54, 43.3)) and diabetes mellitus (OR: 4.10, 95% CI (1.31, 12.4)) were independently associated with stroke. Harboring the NOTCH3 R544C variant in the Taiwan Biobank is comparable with a 6.74 standard deviations increase in individual's polygenic risk score for stroke. CONCLUSION: While the NOTCH3 R544C variant alone increased the risk of stroke, modifiable vascular risk factors also played a role in the occurrence of stroke in Taiwanese community-dwelling individuals carrying the NOTCH3 variant.

Changes in searching behaviour of CSL transcription complexes in Notch active conditions.

During development cells receive a variety of signals, which are of crucial importance to their fate determination. One such source of signal is the Notch signalling pathway, where Notch activity regulates expression of target genes through the core transcription factor CSL. To understand changes in transcription factor behaviour that lead to transcriptional changes in Notch active cells, we have probed CSL behaviours in real time, using in vivo Single Molecule Localisation Microscopy. Trajectory analysis reveals that Notch-On conditions increase the fraction of bound CSL molecules, but also the proportion of molecules with exploratory behaviours. These properties are shared by the co-activator Mastermind. Furthermore, both CSL and Mastermind, exhibit characteristics of local exploration near a Notch target locus. A similar behaviour is observed for CSL molecules diffusing in the vicinity of other bound CSL clusters. We suggest therefore that CSL acquires an exploratory behaviour when part of the activation complex, favouring local searching and retention close to its target enhancers. This change explains how CSL can efficiently increase its occupancy at target sites in Notch-On conditions.

The IRE1/Xbp1 axis restores ER and tissue homeostasis perturbed by excess Notch in Drosophila.

Notch signaling controls numerous key cellular processes including cell fate determination and cell proliferation. Its malfunction has been linked to many developmental abnormalities and human disorders. Overactivation of Notch signaling is shown to be oncogenic. Retention of excess Notch protein in the endoplasmic reticulum (ER) can lead to altered Notch signaling and cell fate, but the mechanism is not well understood. In this study, we show that V5-tagged or untagged exogenous Notch is retained in the ER when overexpressed in fly tissues. Furthermore, we show that Notch retention in the ER leads to robust ER enlargement and elicits a rough eye phenotype. Gain-of-function of unfolded protein response (UPR) factors IRE1 or spliced Xbp1 (Xbp1-s) alleviates Notch accumulation in the ER, restores ER morphology and ameliorates the rough eye phenotype. Our results uncover a pivotal role of the IRE1/Xbp1 axis in regulating the detrimental effect of ER-localized excess Notch protein during development and tissue homeostasis.

Comparison of models for stroke-free survival prediction in patients with CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, which is caused by mutations of the NOTCH3 gene, has a large heterogeneous progression, presenting with declines of various clinical scores and occurrences of various clinical event. To help assess disease progression, this work focused on predicting the composite endpoint of stroke-free survival time by comparing the performance of Cox proportional hazards regression to that of machine learning models using one of four feature selection approaches applied to demographic, clinical and magnetic resonance imaging observational data collected from a study cohort of 482 patients. The quality of the modeling process and the predictive performance were evaluated in a nested cross-validation procedure using the time-dependent Brier Score and AUC at 5 years from baseline, the former measuring the overall performance including calibration and the latter highlighting the discrimination ability, with both metrics taking into account the presence of right-censoring. The best model for each metric was the componentwise gradient boosting model with a mean Brier score of 0.165 and the random survival forest model with a mean AUC of 0.773, both combined with the LASSO feature selection method.

The epigenetics orchestra of Notch signaling: a symphony for cancer therapy.

The aberrant regulation of the Notch signaling pathway, which is a fundamental developmental pathway, has been implicated in a wide range of human cancers. The Notch pathway can be activated by both canonical and noncanonical Notch ligands, and its role can switch between acting as an oncogene or a tumor suppressor depending on the context. Epigenetic modifications have the potential to modulate Notch and its ligands, thereby influencing Notch signal transduction. Consequently, the utilization of epigenetic regulatory mechanisms may present novel therapeutic opportunities for both single and combined therapeutics targeted at the Notch signaling pathway. This review offers insights into the mechanisms governing the regulation of Notch signaling and explores their therapeutic potential.