A Comprehensive Bioinformatic Analysis of NOTCH Pathway Involvement in Stomach Adenocarcinoma

Background. Activation of NOTCH signaling pathways, which are key regulators of multiple cellular functions, has been frequently implicated in cancer pathogenesis, and NOTCH inhibitors have received much recent focus in the context of cancer therapeutics. However, the role and possible involvement of NOTCH pathways in stomach adenocarcinoma (STAD) are unclear. Here, putative regulatory mechanisms and functions of NOTCH pathways in STAD were investigated. Methods. Publicly available data from the TCGA-STAD database were utilized to explore the involvement of canonical NOTCH pathways in STAD by analyzing RNA expression levels of NOTCH receptors, ligands, and downstream genes. Statistical analysis of the data pertaining to cancer and noncancerous samples was performed using R software packages and public databases/webservers. Results. Significant differential gene expression between control and STAD samples was noted for all NOTCH receptors (NOTCH1, 2, 3, and 4), the delta-like NOTCH ligands (DLL-3 and 4), and typical downstream genes (HES1 and HEY1). Four genes (NOTCH1, NOTCH2, NOTCH3, and HEY1) presented prognostic values for the STAD outcome in terms of overall survival. Functional enrichment analysis indicated that NOTCH family genes-strongly correlated genes were mainly enriched in several KEGG signaling pathways such as the PI3K-Akt signaling pathway, human papillomavirus infection, focal adhesion, Rap1 signaling pathway, and ECM-receptor interaction. Gene set enrichment analysis (GSEA) results showed that NOTCH family genes-significantly correlated genes were mainly enriched in four signaling pathways, ECM (extracellular matrix), tumor angiogenesis, inflammatory response, and immune regulation. Conclusions. NOTCH family genes may play an essential role in the progression of STAD by modulating immune cells and mediating ECM synthesis, angiogenesis, focal adhesion, and PI3K-Akt signaling. Multiple NOTCH family genes are valuable candidate biomarkers or therapeutic targets for the management of STAD.

A somatic proteoglycan controls Notch-directed germ cell fate

Communication between the soma and germline optimizes germ cell fate programs. Notch receptors are key determinants of germ cell fate but how somatic signals direct Notch-dependent germ cell behavior is undefined. Here we demonstrate that SDN-1 (syndecan-1), a somatic transmembrane proteoglycan, controls expression of the GLP-1 (germline proliferation-1) Notch receptor in the Caenorhabditis elegans germline. We find that SDN-1 control of a somatic TRP calcium channel governs calcium-dependent binding of an AP-2 transcription factor (APTF-2) to the glp-1 promoter. Hence, SDN-1 signaling promotes GLP-1 expression and mitotic germ cell fate. Together, these data reveal SDN-1 as a putative communication nexus between the germline and its somatic environment to control germ cell fate decisions.

Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes

Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy “maturation arrested state” and resume their natural route of proliferation, hypertrophy, and terminal differentiation. The latter are hallmarks of OA that are easily evaluated in vitro in 2-D or 3-D culture models. The aim of our study was to investigate the effect of NOTCH1 knockdown on proliferation (cell count and Picogreen mediated DNA quantification), cell cycle (flow cytometry), hypertrophy (gene and protein expression of key markers such as RUNX2 and MMP-13), and terminal differentiation (viability measured in 3-D cultures by luminescence assay) of human OA chondrocytes. NOTCH1 silencing of OA chondrocytes yielded a healthier phenotype in both 2-D (reduced proliferation) and 3-D with evidence of decreased hypertrophy (reduced expression of RUNX2 and MMP-13) and terminal differentiation (increased viability). This demonstrates that NOTCH1 is a convenient therapeutic target to attenuate OA progression.

Comprehensive O-GlcNAc glycoproteomics on NOTCH1 EGF repeats refined sequons for O-GlcNAcylation and uncovered unique Lewis X epitopes in mammals

The O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms of O-glycans on epidermal growth factor-like (EGF) repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specific O-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongated O-GlcNAc glycans have not been extensively explored. To extend our view of the specificity and variety of the glycan modification, we conducted a comprehensive analysis of O-GlcNAc glycans on NOTCH1 in mammals. Mass spectrometric analysis of NOTCH1 fragments expressed in HEK293T cells revealed that several EGF domains with putative O-GlcNAcylation sites were hardly modified with O-GlcNAc. Although amino acid residues before the modification site are preferentially occupied with aromatic residues, Phe and Tyr are preferrable to Trp for the apparent modification with O-GlcNAc. Furthermore, a minor form of fucosylated O-GlcNAc glycans was detected in a subset of EGF domains. Fucosylation of O-GlcNAc glycans was enhanced by FUT1, FUT2, or FUT9 expression. The FUT9-dependent Lewis X epitope was confirmed by immunoblotting using an anti-Lewis X antibody. As expected from the similarity in the glycan structures, the Lexis X antigen was detected on O-fucose glycans. Notably, the Lewis X structure on O-glycans was identified in endogenous NOTCH1 isolated from MCF7 cells. Our results refined the putative consensus sequence for the EOGT-dependent extracellular O-GlcNAc modification in mammals and revealed the structural diversity of functional Notch O-glycans.

The Role of Intracellular Trafficking of Notch Receptors in Ligand-Independent Notch Activation

Aberrant Notch signaling has been found in a broad range of human malignancies. Consequently, small molecule inhibitors and antibodies targeting Notch signaling in human cancers have been developed and tested; however, these have failed due to limited anti-tumor efficacy because of dose-limiting toxicities in normal tissues. Therefore, there is an unmet need to discover novel regulators of malignant Notch signaling, which do not affect Notch signaling in healthy tissues. This review provides a comprehensive overview of the current knowledge on the role of intracellular trafficking in ligand-independent Notch receptor activation, the possible mechanisms involved, and possible therapeutic opportunities for inhibitors of intracellular trafficking in Notch targeting.

Mathematical analysis of the effect of portal vein cells on biliary epithelial cell differentiation through the Delta-Notch signaling pathway

Objective The Delta-Notch signaling pathway induces fine-grained patterns of differentiation from initially homogeneous progenitor cells in many biological contexts, including Drosophila bristle formation, where mathematical modeling reportedly suggests the importance of production rate of the components of this signaling pathway. In contrast, the epithelial differentiation of bile ducts in the developing liver is unique in that it occurs around the portal vein cells, which express extremely high amounts of Delta ligands and act as a disturbance for the amount of Delta ligands in the field by affecting the expression levels of downstream target genes in the cells nearby. In the present study, we mathematically examined the dynamics of the Delta-Notch signaling pathway components in disturbance-driven biliary differentiation, using the model for fine-grained patterns of differentiation. Results A portal vein cell induced a high Notch signal in its neighboring cells, which corresponded to epithelial differentiation, depending on the production rates of Delta ligands and Notch receptors. In addition, this epithelial differentiation tended to occur in conditions where fine-grained patterning was reported to be lacking. These results highlighted the potential importance of the stability towards homogeneity determined by the production rates in Delta ligands and Notch receptors, in a disturbance-dependent epithelial differentiation.

Notch receptors in GtoPdb v.2021.2

Aberrant Notch signalling is implicated in a number of human cancers [18, 26, 13, 21], and there is intense pharmaceutical activity being directed towards achieving clinically effective Notch pathway inhibition [7, 16].

A Comprehensive Bioinformatics Analysis of Notch Pathways in Bladder Cancer

Background: A hallmark of Notch signaling is its variable role in tumor biology, ranging from tumor-suppressive to oncogenic effects. Until now, the mechanisms and functions of Notch pathways in bladder cancer (BCa) are still unclear. Methods: We used publicly available data from the GTEx and TCGA-BLCA databases to explore the role of the canonical Notch pathways in BCa on the basis of the RNA expression levels of Notch receptors, ligands, and downstream genes. For statistical analyses of cancer and non-cancerous samples, we used R software packages and public databases/webservers. Results: We found differential expression between control and BCa samples for all Notch receptors (NOTCH1, 2, 3, 4), the delta-like Notch ligands (DLL1, 3, 4), and the typical downstream gene hairy and enhancer of split 1 (HES1). NOTCH2/3 and DLL4 can significantly differentiate non-cancerous samples from cancers and were broadly altered in subgroups. High expression levels of NOTCH2/3 receptors correlated with worse overall survival (OS) and shorter disease-free survival (DFS). However, at long-term (>8 years) follow-up, NOTCH2 expression was associated with a better OS and DFS. Furthermore, the cases with the high levels of DLL4 were associated with worse OS but improved DFS. Pathway network analysis revealed that NOTCH2/3 in particular correlated with cell cycle, epithelial–mesenchymal transition (EMT), numbers of lymphocyte subtypes, and modulation of the immune system. Conclusions: NOTCH2/3 and DLL4 are potential drivers of Notch signaling in BCa, indicating that Notch and associated pathways play an essential role in the progression and prognosis of BCa through directly modulating immune cells or through interaction with cell cycle and EMT.

POS0042 NOTCH 1 INHIBITION INCREASES OSTEOCLAST PROGENITOR ACTIVITY IN THE MOUSE MODEL OF RHEUMATOID ARTHRITIS

Background:Osteoclasts mediate periarticular and systemic bone loss in rheumatoid arthritis (RA). Osteoclast progenitor cells (OCPs) derived from the myeloid lineage are susceptible to regulation through Notch signaling. Murine bone marrow and splenic OCPs, identified as CD45+Ly6G-CD3-B220-NK1.1-CD11blo/+CD115+CCR2+ cells, are specifically increased in arthritis. We previously identified an increased frequency of OCPs expressing Notch receptors in arthritic mice.Objectives:Several studies suggested that Notch signaling modulation affects the course of experimental arthritis. We aimed to determine the effects of Notch receptor signaling inhibition on OCP activity and arthritis severity in murine collagen-induced arthritis (CIA).Methods:Male C57/Bl6 and DBA mice were immunized with chicken type II collagen and treated with i.p. injections of anti-Notch 1 neutralizing antibodies (1mg/kg). Notch receptor 1 through 4 expression on OCPs was analyzed by flow cytometry in periarticular bone marrow (PBM) and spleen (SPL). Gene expression of Notch receptors, ligands and transcription targets as well as osteoclast differentiation genes RANK, cFos and cFms was determined by qPCR from tissues and sorted OCPs. FACS sorted OCPs were stimulated by osteoclastogenic factors (M-CSF and RANKL), in control, IgG, Jagged (Jag)1 or Delta-like (DLL)1 coated wells, with or without anti-Notch 1 antibodies. Research was approved by the Ethics Committee.Results:We confirmed the expression of Notch receptors on OCPs by flow cytometry with Notch 1 and 2 being most abundantly expressed (around 25% and 40% positive OCPs in PBM and 35% and 20% in SPL respectively), with a significant increase of Notch 2 expression in arthritis. Seeding OCPs on DLL1 coated wells significantly increased while seeding on Jag1 coated wells significantly decreased osteoclastogenesis as reflected on the number of TRAP+ osteoclasts and expression of osteoclast differentiation genes. The addition of anti-Notch 1 antibodies to ligand-stimulated OCPs resulted in an increased number of TRAP+ osteoclasts, partially reversing Jag1 inhibition. In vivo treatment with anti-Notch 1 antibodies did not affect total OCP frequency, but increased expression of Notch 4 both in PBM and SPL as seen by flow cytometry and qPCR. Additionally, anti-Notch 1 treatment stimulated Notch transcription factors HES and HEY. Both PBM and SPL cultured OCPs from anti-Notch 1 treated mice produced a higher number of large TRAP+ osteoclasts, doubling the area covered with osteoclasts in the latter compared to untreated mice. Increased osteoclastogenesis in vitro was further confirmed by an increased expression of osteoclast differentiation genes in the treated group.Conclusion:Our results confirm that Notch signaling may represent an important therapeutic target for the regulation of osteoclast activity in arthritis. Both in vitro and in vivo anti-Notch 1 neutralizing antibodies enhanced osteoclastogenesis in CIA model, implying an inhibitory role of Notch 1 signaling in osteoclast differentiation. As Notch 2 expression is increased on OCPs of arthritic mice, we next plan to determine the effects of Notch 2 neutralization on osteoclast activity and arthritis severity.References:[1]Ikić Matijašević M, Flegar D, Kovačić N, Katavić V, Kelava T, Šućur A, et al. Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis. Clin Exp Immunol. 2016;186(3):321–35.[2]Šućur A, Filipović M, Flegar D, Kelava T, Šisl D, Lukač N, et al. Notch receptors and ligands in inflammatory arthritis – a systematic review. Immunology Letters 2020 Vol. 223, p. 106–14.Acknowledgements:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, UIP-2017-05-1965 and DOK-2018-09-4276.Disclosure of Interests:None declared.

JAG1 intracellular domain acts as a transcriptional cofactor that forms an oncogenic transcriptional complex with DDX17/SMAD3/TGIF2

Jagged1 (JAG1) is a Notch ligand that contact-dependently activates Notch receptors and regulates cancer progression. The JAG1 intracellular domain (JICD1) is generated from JAG1, such as the formation of NOTCH1 intracellular domain (NICD1), however, the role of JICD1 in tumorigenicity has not been comprehensively elucidated. Herein, we revealed that JICD1 induced astrocytes to acquire several cancer stem cell properties, including tumor formation, invasiveness, stemness, and resistance to chemotherapy and radiotherapy. The transcriptome, ChIP-sequencing, and proteomic analyses revealed that JICD1 increased SOX2 expression by forming a transcriptional complex with DDX17, SMAD3, and TGIF2. Furthermore, JICD1-driven tumorigenicity was directly regulated by SOX2. Therefore, our results demonstrated that, like NICD1, JICD1 acts as a transcriptional cofactor in the formation of the DDX17/SMAD3/TGIF2 transcriptional complex, leading to oncogenic transformation.