Jagged1 Research Articles

Multiple myeloma exploits Jagged1 and Jagged2 to promote intrinsic and bone marrow-dependent drug resistance.

Multiple myeloma is still incurable due to an intrinsic aggressiveness or, more frequently, to the interactions of malignant plasma cells with the bone marrow (BM) microenvironment. Myeloma cells educate BM cells to support neoplastic cell growth, survival, acquisition of drug resistance resulting in disease relapse. Myeloma microenvironment is characterized by Notch signaling hyperactivation due to the increased expression of Notch1 and 2 and the ligands Jagged1 and 2 in tumor cells. Notch activation influences myeloma cell biology and promotes the reprogramming of BM stromal cells. In this work we demonstrate, in vitro, ex vivo and by using a zebrafish multiple myeloma model, that Jagged inhibition causes a decrease in both myeloma-intrinsic and stromal cell-induced resistance to currently used drugs, i.e. bortezomib, lenalidomide and melphalan. The molecular mechanism of drug resistance involves the chemokine system CXCR4/SDF1α. Myeloma cell-derived Jagged ligands trigger Notch activity in BM stromal cells. These, in turn, secrete higher levels of SDF1α in the BM microenvironment increasing CXCR4 activation in myeloma cells, which is further potentiated by the concomitant increased expression of this receptor induced by Notch activation. Consistently with the augmented pharmacological resistance, SDF1α boosts the expression of BCL2, Survivin and ABCC1. These results indicate that a Jagged-tailored approach may contribute to disrupting the pharmacological resistance due to intrinsic myeloma cell features or to the pathological interplay with BM stromal cells and, conceivably, improve patients’ response to standard-of-care therapies.

Association of JAG1 gene polymorphism with systemic blood pressure in patients with obstructive sleep apnea: a prospective cohort study

AimTo assess the effects of single nucleotide polymorphisms (SNPs) on blood pressure control in patients with obstructive sleep apnea (OSA).MethodsThis prospective observational cohort study, conducted between 2004 and 2014, examined the associations of SNPs of JAG1, GUCY1A3-GUCY1B3, SH2B3, and NPR3-C5orf23 genes with systolic and diastolic blood pressure (SBP, DBP) in 1179 adults evaluated for OSA with overnight polysomnography. Genotyping was performed by unlabeled probe melting analysis.ResultsThe patients were predominantly male (69.6%, mean age 52 ± 11 years, apnea-hypopnea index 34 ± 31 episodes/h). Only JAG1 genotype was associated with SBP and DBP: compared with AA homozygotes, G allele carriers (pooled GG and AG genotype) had significantly higher morning SBP (132 ± 19 vs 129 ± 18 mm Hg; P = 0.009) and morning and evening DBP (85 ± 11 vs 83 ± 10 mm Hg, P = 0.004; 86 ± 10 vs 84 ± 10 mm Hg, P = 0.012, respectively); the differences remained significant after the correction for multiple SNPs testing. In multivariate analyses, oxygen desaturation index and JAG1 genotype independently predicted morning SBP (P = 0.001, P = 0.003, respectively) and DBP (P < 0.001, P = 0.005, respectively), and evening SBP (P = 0.019, P = 0.048, respectively) and DBP (P = 0.018, P = 0.018, respectively).ConclusionThis is the first replication study of the SNPs recently linked to arterial hypertension in general population by genome-wide association studies. Our findings suggest that JAG1 genotype is related to blood pressure control in OSA: G allele was associated with higher morning and evening SBP and DBP.

Novel mutations in NOTCH2 gene in infants with neonatal cholestasis.

One cause of neonatal cholestasis (NC) is paucity of intrahepatic bile ducts which can be associated with Alagille syndrome or non- syndromic. Alagille syndrome is caused by autosomal dominant mutations in the Notch signaling pathway ligand Jagged1 in 94% of patients and mutations in the NOTCH2 receptor in <1% of patients. This is a retrospective case series studying infants with neonatal cholestasis found to have variants of unknown significance (VOUS) in NOTCH2. Sorting intolerant from tolerant (SIFT) and polymorphism phenotyping (PolyPhen) were utilized to predict a damaging effect. Five infants with NC without other features of Alagille syndrome were found to have one copy of a VOUS in NOTCH2, predicted to be damaging by SIFT and PolyPhen. Our cases support the notion that NOTCH2 mutations may result in hypoplastic biliary system. Further characterization of these variants is important to assist with our clinical approach to NC.

Alagille syndrome mutation update: Comprehensive overview of JAG1 and NOTCH2 mutation frequencies and insight into missense variant classification.

Alagille syndrome is an autosomal dominant disease with a known molecular etiology of dysfunctional Notch signaling caused primarily by pathogenic variants in JAGGED1 (JAG1), but also by variants in NOTCH2. The majority of JAG1 variants result in loss of function, however disease has also been attributed to lesser understood missense variants. Conversely, the majority of NOTCH2 variants are missense, though fewer of these variants have been described. In addition, there is a small group of patients with a clear clinical phenotype in the absence of a pathogenic variant. Here, we catalog our single‐center study, which includes 401 probands and 111 affected family members amassed over a 27‐year period, to provide updated mutation frequencies in JAG1 and NOTCH2 as well as functional validation of nine missense variants. Combining our cohort of 86 novel JAG1 and three novel NOTCH2 variants with previously published data (totaling 713 variants), we present the most comprehensive pathogenic variant overview for Alagille syndrome. Using this data set, we developed new guidance to help with the classification of JAG1 missense variants. Finally, we report clinically consistent cases for which a molecular etiology has not been identified and discuss the potential for next generation sequencing methodologies in novel variant discovery.

Inhibitor of γ-secretase alleviates middle ear inflammation by regulating Th2 response in OVA-mediated allergic OME in vivo.

Otitis media with effusion (OME) often occurs in infants and young children, which is related to allergic reactions. Notch signaling pathway plays an important role in allergic responses. In this study, we aimed to investigate the role of Notch signaling pathway in the ovalbumin (OVA)-mediated allergic OME in vivo. OVA-induced OME rats were treated with a control vehicle or a γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) suppressing the Notch signaling. We studied the effect of Notch signaling pathway in OME model, including histopathological assessment, the expression of Th1 cytokines (IFN-γ), Th2 cytokines (IL-4, IL-5), key transcription factors (T-bet, GATA-3) by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the level of Notch ligand (Jagged1) and the downstream target gene Hes1 were also evaluated by qRT-PCR and immunofluorescent staining. We observed that the production of Th2 cytokines was increased, the level of Th1 cytokines was decreased in OME experimental model. Likewise, Th2-cytokine(IL-4)level was reduced, but the level of Th1 cytokines(IFN-γ) was no changes. Additionally, administration of DAPT induced a decrease in the expression of GATA-3 mRNA, however, no influence on T-bet mRNA production. These results suggest that there is an imbalance with Th1/Th2 in OVA-mediated allergic OME. DAPT treatment can block the Notch signaling pathway and relieve the middle ear inflammation through modulating the level of Th2 responses in OVA-induced allergic OME.

Cystic biliary atresia with paucity of bile ducts and gene mutation in KDM6A: a case report

BackgroundBiliary atresia (BA) cases are generally not associated with congenital abnormalities. However, accurate diagnosis of BA is often challenging because the histopathological features of BA overlap with those of other pediatric liver diseases and rarely overlap with those of other genetic disorders. We experienced a rare case of BA with the histopathological finding of bile duct paucity, a gene mutation in KDM6A, and KS-like phenotypes.Case presentationA male baby was diagnosed with biliary atresia by intraoperative cholangiography at 4 days of age, and histological examination following a liver biopsy revealed a paucity of bile ducts and several typical clinical findings of Alagille syndrome. However, Alagille syndrome was ruled out after neither JAG1 nor NOTCH2 gene mutations were identified. Whole-exome sequencing on DNA from his parents was additionally performed to examine other possible syndromic disorders, and a mutation was identified in KDM6A. However, Kabuki syndrome was not diagnosed as a result.The histological finding of interlobular bile duct paucity and the genetic mutation in KDM6A, as well as several clinical findings consistent with Alagille syndrome or Kabuki syndrome, made it difficult to confirm the diagnosis of BA.ConclusionsBased on the interesting findings of the present case, we hypothesized that KDM6A is associated with hepatic malformations via a connection with the Notch signaling pathway.

Tbx1 and Foxi3 genetically interact in the pharyngeal pouch endoderm in a mouse model for 22q11.2 deletion syndrome.

We investigated whether Tbx1, the gene for 22q11.2 deletion syndrome (22q11.2DS) and Foxi3, both required for segmentation of the pharyngeal apparatus (PA) to individual arches, genetically interact. We found that all Tbx1+/-;Foxi3+/- double heterozygous mouse embryos had thymus and parathyroid gland defects, similar to those in 22q11.2DS patients. We then examined Tbx1 and Foxi3 heterozygous, null as well as conditional Tbx1Cre and Sox172A-iCre/+ null mutant embryos. While Tbx1Cre/+;Foxi3f/f embryos had absent thymus and parathyroid glands, Foxi3-/- and Sox172A-iCre/+;Foxi3f/f endoderm conditional mutant embryos had in addition, interrupted aortic arch type B and retroesophageal origin of the right subclavian artery, which are all features of 22q11.2DS. Tbx1Cre/+;Foxi3f/f embryos had failed invagination of the third pharyngeal pouch with greatly reduced Gcm2 and Foxn1 expression, thereby explaining the absence of thymus and parathyroid glands. Immunofluorescence on tissue sections with E-cadherin and ZO-1 antibodies in wildtype mouse embryos at E8.5-E10.5, revealed that multilayers of epithelial cells form where cells are invaginating as a normal process. We noted that excessive multilayers formed in Foxi3-/-, Sox172A-iCre/+;Foxi3f/f as well as Tbx1 null mutant embryos where invagination should have occurred. Several genes expressed in the PA epithelia were downregulated in both Tbx1 and Foxi3 null mutant embryos including Notch pathway genes Jag1, Hes1, and Hey1, suggesting that they may, along with other genes, act downstream to explain the observed genetic interaction. We found Alcam and Fibronectin extracellular matrix proteins were reduced in expression in Foxi3 null but not Tbx1 null embryos, suggesting that some, but not all of the downstream mechanisms are shared.

Delta-like 1-mediated cis-inhibition of Jagged1/2 signalling inhibits differentiation of human epidermal cells in culture

Epidermal homeostasis depends on a balance between self-renewal of stem cells and terminal differentiation of their progeny. Notch signalling is known to play a role in epidermal stem cell patterning and differentiation. However, the molecular mechanisms are incompletely understood. Here we demonstrate dynamic patterns of Notch ligand and receptor expression in cultured human epidermis. Notch2 and 3 act together to promote differentiation, while Notch1 decreases stem cell proliferation. The Notch ligand Jagged1 triggers differentiation when presented on an adhesive substrate or on polystyrene beads and over-rides the differentiation inhibitory effect of cell spreading. In contrast, Delta-like 1 (Dll1) overexpression abrogates the pro-differentiation effect of Jagged1 in a cell autonomous fashion. We conclude that Dll1 expression by stem cells not only stimulates differentiation of neighbouring cells in trans, but also inhibits differentiation cell autonomously. These results highlight the distinct roles of different Notch receptors and ligands in controlling epidermal homeostasis.

MiR-449a regulates insulin signalling by targeting the Notch ligand, Jag1 in skeletal muscle cells

BackgroundmiR-449a, an intronic miRNA, is highly down-regulated in the skeletal muscle during diabetes. Its levels are epigenetically regulated by altered acetylation/deacetylation on the promoter that it shares with its host gene, Cdc20b. However, the cellular role of this epigenetically regulated miRNA in the muscle during diabetes is not well understood. Here, we sought to unravel the crosstalk between altered miR-449a expression and impaired skeletal muscle metabolism.MethodsPredicted targets of miR-449a were extracted using online available target prediction tools. Differentiated C2C12 cells were transfected with the miR-449a mimic and/or its inhibitor and the levels of the target mRNA and protein was evaluated by qRT-PCR and Western Blot analysis. This was validated by luciferase wild type and mutated constructs of the target 3’UTR. Inhibition of Notch signalling was assessed by evaluating the transcript levels of Notch target genes, Hes1 and Hey1 and the status of NICD (Notch Intracellular domain) by immunofluoresence microscopy. Effect of miR-449a on insulin signalling was evaluated by monitoring insulin induced PI3K and AKT phosphorylation and glucose uptake.ResultsOur data demonstrate that in C2C12 skeletal muscle cells, miR-449a binds to the 3’UTR of Jag1, an important Notch ligand, and down-regulates, both its transcript and protein levels. This was, however, prevented in the presence of the miR-449a inhibitor that suggests the specificity of the miRNA effect. This was validated in human primary skeletal muscle cells where miR-449a decreased Jag1 protein levels and this was prevented in the presence of the miR-449a inhibitor. This miR-449a-Jag1 interaction subsequently affects the Notch signalling pathway as was evident by the fact that miR-449a decreased the levels of NICD and consequently, the levels of Notch target genes, Hes1 and Hey1 were significantly inhibited. miR-449a and Notch pathway inhibition using DAPT, significantly increased insulin stimulated PI3K and AKT phosphorylation and these were prevented in the presence of the miR-449a inhibitor.ConclusionOur results indicate towards a critical role for miR-449a and its target, Jag1 in regulating Notch signalling and insulin signalling in the skeletal muscle and imply that targeting this axis might hold therapeutic potential for impaired skeletal muscle metabolism during diabetes.

Oxysterol/chitotriosidase based selective screening for Niemann-Pick type C in infantile cholestasis syndrome patients

BackgroundNiemann-Pick disease type C (NP-C) is an inherited neurodegenerative disease (1 per 100 000 newborns) caused by NPC proteins impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. To date the NP-C diagnostics is usually based on cholesterol detection in fibroblasts using an invasive and time-consuming Filipin staining and we need more arguments to widely introduce oxysterols as a biomarkers in NP-C.MethodsInsofar as NP-C represents about 8% of all infant cholestases, in this prospective observational study we tried to re-assess the specificity plasma oxysterol and chitotriosidase as a biochemical screening markers of NP-C in children with cholestasis syndrome of unknown origin. For 108 patients (aged from 2 weeks to 7 years) the levels of cholestane-3β,5α,6β-triol (C-triol) and chitotriosidase (ChT) were measured. For patients with elevated C-triol and/or ChT the NPC1 and NPC2 genes were Sanger-sequenced and 47 additional genes (from the custom liver damage panel) were NGS-sequenced.ResultsIncreased C-triol level (> 50 ng/ml) was detected in 4 (of 108) infants with cholestasis syndrome of unknown origin, with following molecular genetic NP-C diagnosis for one patient. Plasma cholesterol significantly correlates with C-triol (p < 0.05). NGS of high C-triol infants identified three patients with mutations in JAG1 (Alagille syndrome) and ABCB11 (Byler disease) genes. Increased ChT activity was detected in 8 (of 108) patients with various aetiologies, including NP-C, Byler disease and biliary atresia.ConclusionCombined analysis of ChT activity and C-triol levels is an effective method for identifying NP-C.

Notch signaling regulates osteosarcoma proliferation and migration through Erk phosphorylation

We used a murine spontaneous osteosarcoma cell line with high metastatic potential, the K7M2 cell line to study the role of Notch signaling in the biological manifestations of osteosarcoma, to understand its underlying mechanism in the regulation of cell proliferation and migration, and to improve patient prognosis in cases of osteosarcoma through the discovery of novel therapeutic targets, First, Notch expression in K7M2 was determined by immunostaining, and the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was used to inhibit proteolytic cleavage of the Notch intracellular domain (NICD), resulting in the inhibition of Notch activation. By using the Sulforhodamine B assay, colony-forming units assay, Brdu and Ki67 staining, and flow cytometry assays of apoptosis and cell cycle stage, DAPT was found to inhibit K7M2 proliferation in a dose-dependent manner. By using wound healing and transwell migration assays, DAPT was found to inhibit K7M2 migration in a dose-dependent manner as well. By using a combination of micro-Raman spectroscopy and K-means clustering analysis, we found that DAPT inhibit a variety of important cell metabolism-related components in most K7M2 cell structures. Then, DAPT was found to inhibit Notch1ICD expression in a concentration-dependent manner, and this expression was directly correlated with Phospho-Erk1/2 (p-Erk) by using Western blotting. To confirm this finding, we used the Notch signaling ligand Jagged1 to activate the Notch signaling pathway, which in turn up-regulated p-Erk, resulting in increased proliferation and migration of K7M2. Using the Erk pathway inhibitor U0126, we showed that p-Erk was downregulated and the proliferation and migration of K7M2 decreased along with it. Finally, we constructed a K7M2 mouse para-tibial tumor model and lung metastatic model. We found DAPT inhibits p-Erk in vivo, effectively controls tumor growth, reduces angiogenesis, reduces metastasis to the lungs, and improves overall survival. In summary, Notch signaling plays an oncogene role and promotes metastasis in osteosarcoma through p-Erk. DAPT effectively inhibits osteosarcoma proliferation and metastasis in vivo and in vitro by inhibiting Erk phosphorylation. Therefore, the inhibition of Notch activation resulted the down-regulation of phosphorylation of Erk pathway can be used as potential therapeutic targets in clinical treatment to improve osteosarcoma prognosis.

Downregulation of Notch Signaling in Kras-Induced Gastric Metaplasia

Activating mutations and amplification of Kras and, more frequently, signatures for Kras activation are noted in stomach cancer. Expression of mutant KrasG12D in the mouse gastric mucosa has been shown to induce hyperplasia and metaplasia. However, the mechanisms by which Kras activation leads to gastric metaplasia are not fully understood. Here we report that KrasLSL-G12D/+;Pdx1-cre, a mouse model known for pancreatic cancer, also mediates KrasG12D expression in the stomach, causing gastric hyperplasia and metaplasia prior to the pathologic changes in the pancreas. These mice exhibit ectopic cell proliferation at the base of gastric glands, whereas wild-type mice contain proliferating cells primarily at the isthmus/neck of the gastric glands. Notch signaling is decreased in the KrasLSL-G12D/+;Pdx1-cre gastric mucosa, as shown by lower levels of cleaved Notch intracellular domains and downregulation of Notch downstream target genes. Expression of a Notch ligand Jagged1 is downregulated at the base of the mutant gland, accompanied by loss of chief cell marker Mist1. We demonstrate that exogenous Jagged1 or overexpression of Notch intracellular domain stimulates Mist1 expression in gastric cancer cell lines, suggesting positive regulation of Mist1 by Notch signaling. Finally, deletion of Jagged1 or Notch3 in KrasLSL-G12D/+;Pdx1-cre mice promoted development of squamous cell carcinoma in the forestomach, albeit short of invasive adenocarcinoma in the glandular stomach. Taken together, these results reveal downregulation of Notch signaling and Mist1 expression during the initiation of Kras-driven gastric tumorigenesis and suggest a tumor-suppressive role for Notch in this context.

Abstract 3439: Sulindac sulfide as a gamma secretase modifier to target triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is defined as pathologically negative for estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 amplification (HER2-). TNBCs are a heterogeneous group of clinically aggressive breast cancers with high risk of recurrence and metastasis, but the current treatment options remain limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, Including Gamma Secretase Inhibitors (GSIs) are quite effective in preclinical models of TNBC. However the success of GSIs in clinical trials is limited by their intestinal toxicity and adverse immunological effects. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects. We identified Sulindac Sulfide (SS), the active metabolite of FDA-approved NSAID Sulindac, as a potential candidate to replace GSIs. SS has documented Gamma Secretase Modifier (GSM) activity, in addition to cyclo-oxygenase (COX) inhibition. We confirmed that SS inhibits Notch1 cleavage in TNBC cells, but not in murine T-cells. SS significantly inhibited mammospheres growth in all human and murine TNBC models we tested: 1) human MDA-MB-231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe (LFng-/-); and 3) Two TNBC patient-derived xenograft models, 2K1 and 4IC. In C0321 tumors, we found that SS had remarkable single-agent anti-tumor activity and virtually eliminated Notch1 expression in tumors. SS caused an increase in intra-tumoral CD11c+ dendritic cells, but decreased CD4 cells, which in this model are largely PD-1 positive (exhausted). CD8 cells were modestly increased. SS did not affect the numbers of tumor infiltrating macrophages or myeloid-derived suppressor cells (MDSC). However, SS blocked the immunosuppressive function of bone marrow-derived MDSC. RNA-Sequencing of SS-treated tumors revealed significant reduction of CXCL14, EGR1, HOXC6, MAGI2, NCAM1, APOE, CLU (a Wnt target), DTX4 (an E3-ligase positive regulator of Notch activation), and TGFB3 genes and upregulation of CCL17, EPCAM, FABP4, C4A, LTF, ZBTB16, INADL, and FGFR2 genes. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be significantly easier and more cost-effective than developing unproven investigational agents. Citation Format: Fokhrul Hossain, Deniz A Ucar, Samarpan Majumder, Margarite Matossian, Keli Xu, Yong Ran, Lisa Minter, Yaguang Xi, Matthew Burow, Todd Golde, Barbara Osborne, Lucio Miele. Sulindac sulfide as a gamma secretase modifier to target triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3439.

Abstract 4623: Notch3 in lung adenocarcinoma pathogenesis and heterogeneity

Abstract Lung cancer is the leading cause of cancer related death worldwide. Non-small lung cancer accounts for 85% of diagnoses and the predominant subtype is lung adenocarcinoma (LUAD). LUAD has a less than 20% response rate to conventional chemotherapy and despite identification of several therapeutic targets, the majority of diagnoses continue to lead to death, highlighting the need for better treatments. Notch signaling in mammals is complex, with multiple receptors, ligands and modifiers. Notch ligands at the surface of one cell interact with receptors on an adjacent cell, triggering cleavage and nuclear translocation of the intracellular domain which interacts with Rbpj/Csl to control the transcription of target genes. In LUAD, significant evidence suggests that Notch plays an oncogenic role. Previously, we identified a population of Tumor Propagating Cells (TPC), which had greater capacity for tumor formation and are enriched after treatment with cisplatin. The TPC population was defined by the expression of CD24, ITGB4 and was Notch high. Further functional validation showed that specifically Notch3 contributes to this propagation phenotype and is key in establishing functional intra-tumoral heterogeneity. However, the TPC population is still heterogeneous and likely only a subset of these cells are true TPCs. Furthermore, the gene expression signature of the mouse TPCs has prognostic significance in human LUAD, however the TPC population hasn’t been defined in human patients, limiting our ability to target the Notch pathway therapeutically. To assess the heterogeneity of the mouse TPC population we performed FACS on tumors from KrasLSLG12D; Tp53flox/flox; YFPLSL twelve weeks after tumor initiation via AdCre. Tumors were sorted for bulk population (YFP+Lin-) and for TPC (YFP+Lin-CD24+ITGB4+Notchhi) population and single cell sequencing was performed using the 10X genomics platform. We hypothesize that aberrant Notch3 signaling is a critical pathway for survival in at least some LUAD and that Notch activation leads to gene expression changes that are important for driving human LUAD maintenance. Patient-derived xenografts (PDX) more closely recapitulate important aspects of human cancer, particularly with regards to the role of intratumoral heterogeneity and self-renewal pathways. To characterize Notch3 expressing cells in human LUAD, we performed FACS on our cohort of LUAD PDXs and sorted the Notch3+ population. We found expression of Notch3 and expression of Notch ligand Jagged2 in all LUAD tumors regardless of driver mutation. Citation Format: Kieren D. Marini, Stanley Leung, Alex Lee, Alejandro Sweet-Cordero. Notch3 in lung adenocarcinoma pathogenesis and heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4623.

The Contribution of Gene Mutations to the Pathogenesisof Tetralogy of Fallot

Congenital heart disease (CHD) is considered as an important and developing area in the medical community. Since these patients can reach maturity and have children, the role of genetic determinants in increasing risk of CHD is extremely evident among children of these patients. Because genetic studies related to CHD are increasing, and each day the role of new genetic markers is more and more clarified, this review re-examined the effects of gene mutations in the pathogenesis of tetralogy of Fallot (TOF) as an important pathological model among other CHDs. Due to the complexity of heart development, it is not astonishing that numerous signaling pathways and transcription factors, and many genes are involved in pathogenesis of TOF. This review focuses on the jag1, nkx2.5, gata4, zfpm2/fog2 and cited2 genes previously reported to be involved in TOF.

Knockdown of DANCR reduces osteoclastogenesis and root resorption induced by compression force via Jagged1

ABSTRACTLncRNA DANCR has been proven to be involved in osteoblast differentiation. This study aims to investigate the role of DANCR in osteoclast formation and root resorption in periodontal ligament (PDL) cells induced by compression force (CF). Rat orthodontic tooth movement (OTM) model was established. The molecules expressions in the areas of root resorption form OTM model were measured. The number of osteoclasts was measured using Tartrate-resistant acid phosphatase (TRAP) staining. The bone resorption was detected using pit formation assay. We showed that the expression of DANCR and Jagged1 protein was increased in rat OTM model and human periodontal ligament (hPDL) cells treated with CF, and CF increased the production of Jagged1, RANKL, and IL-6 from the hPDL cells. Moreover, DANCR could positively regulate Jagged1 protein expression. Knockdown of DANCR could change the promotion effect of CF on osteoclastogenesis and bone resorption in vitro and in vivo experiments, while overexpression of Jagged1 reversed si-DANCR effect. Taken together, knockdown of DANCR reduced osteoclast formation and root resorption induced by CF via Jagged1.

The Notch system during pubertal development of the bovine mammary gland

The development of the mammary gland of cows during pre-weaning and puberty will condition its future productive capacity and warrants special study. In this respect, Notch signaling regulates tissue development and fate by modifying cell proliferation and differentiation and has been involved in stem cell maintenance, but has not been extensively studied in the developing mammary glands in cows. We therefore investigated Notch receptor expression and localization, as well as the expression of Notch ligands and target genes in the mammary gland of Holstein heifers in pre- and post-pubertal stages. Notch receptors 1 to 4 were detected by immunohistochemistry in the parenchyma and stroma of the developing gland. The subcellular localization of the four receptors was predominantly cytoplasmic except for NOTCH4, which was mostly nuclear. The membrane and the active intracellular domains of NOTCH paralogues were identified by western blot. NOTCH1 and NOTCH2 active domains increased during pubertal stages while NOTCH3 and NOTCH4 active domains decreased, suggesting strikingly different involvement of NOTCH paralogues in bovine mammary gland development and differentiation. The mRNA expression levels of the target genes HEY1 and HEY2 increased during peri-puberty whereas no variation of HES1 mRNA levels was observed. The mRNA levels of the Notch ligands JAGGED1 and DELTA1 also increased gradually during development. In conclusion, Notch signaling system dynamically varies throughout the development of the mammary gland during puberty pointing to specific time involvement of each component.

Mutual regulation of JAG2 and PRAF2 promotes migration and invasion of colorectal cancer cells uncoupled from epithelial\u2013mesenchymal transition

BackgroundOur previous studies revealed that Jagged 2 (JAG2) is involved in the regulation of migration and invasion of colon cancer cells without affecting cell proliferation. This study further explored the specific mechanism by which JAG2 promotes migration and invasion of colorectal cancer cells.MethodsJAG2 mRNA expression in different clinical stages of colorectal cancer and normal intestinal tissues was detected by quantitative PCR (QPCR). QPCR and Western Blot were used to analyze the differential expression of JAG2 mRNA and protein between normal human colon tissue cells and various colorectal cancer cells. Co-expression status of JAG2 and epithelial–mesenchymal transition (EMT) markers in colon cancer tissues and cells was analyzed. The difference between TGF-β-induced EMT model and the JAG2 overexpression model were compared in promoting migration and invasion of HT29 cells. HT29 cells were treated with EMT pathway inhibitors (LY2157299 and Slug siRNA) to identify a cross-talk between the JAG2 effect and the Notch pathway. Co-expressed genes of JAG2 in colorectal cancer cells were identified using siRNA and transcriptome microarray technology. The mutual regulation of JAG2 and the co-expressed gene PRAF2 and the regulation of the paracrine effect of exosomes were analyzed.ResultsJAG2 was abnormally expressed in colorectal cancer tissues and directly related to clinical stages. Similar to the findings in tissues, the expression of both JAG2 mRNA and protein was significantly increased in the colorectal cancer cell lines compared with that of normal colorectal cell line CCD18-Co. It was shown in our cell model that JAG2 was involved in the regulation of migration and invasion independent of the canonical Notch signaling pathway. More interestingly, JAG2 also promoted the migration and invasion of colon cancer cells in a non-EMT pathway. Further analysis revealed the co-expression of JAG2 with PRAF2 in colorectal cancer cells. JAG2-rich exosomes were released from colorectal cancer cells in a PRAF2-dependent way, while these exosomes regulated the metastasis of colorectal cancer cells in a paracrine manner.ConclusionsThis is the evidence supporting the biological function of JAG2 through non-canonical Notch and non-EMT-dependent pathways and also the first demonstration of the functions of PRAF2 in colorectal cancer cells. These findings also provide theoretical basis for the development of small molecules or biological agents for therapeutic intervention targeting JAG2/PRAF2.

PB1882 DECONSTRUCTING RICHTER SYNDROME TUMOR NICHE: JAGGED‐1 LIGAND LEADS THE WAY

Background:Richter Syndrome (RS) is defined as the occurrence of a high‐grade lymphoma (usually diffuse large B‐cell lymphoma ‐ DLBCL) in patients with a previous/concomitant diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL).RS usually occurs in lymph‐nodes, but sometimes may infiltrate extranodal sites.Isolated cardiac involvement is very unique.Most DLBCL‐type RS does not share lesions with de novo subtypes.NOTCH1 mutation was reported in almost 30% of cases,but NOTCH1 signaling activation independent of its mutational status,as NOTCH ligands mapping in tumor niche, remain scarcely estimated, as opposed to CLL.This has directed our attention to the role of cell non‐autonomous NOTCH1 activation at the cross‐roads between other pathways coming from the niche (i.e. immune checkpoint axis). CLL cells also express the NOTCH ligand JAGGED1 (JAG1), that contributes to CLL pathogenesis by activating its own signaling.Experimental proofs in other settings were made, linking the NOTCH1‐JAG1 signaling to the immunological niche.Strikingly, DLBCL‐type RS exhibits PD‐1/PD‐L1 axis upregulation in up to 80% of cases.Taking advantage of these findings, we decided to perform immunohistochemistry (IHC), using validated antibodies,to dissect the precise anatomy of the NOTCH/ligand interface in RS,in vivo.Aims:1) To investigate context‐dependent, cell‐autonomous/cell non‐autonomous NOTCH1 network activation, using IHC stains. Specifically, we focused on NOTCH1 and its ligand JAG1, a well‐known pivotal player in embryonic heart and cardiac diseases. 2) To zoom into the PD‐1/PDL‐1 axis activation in immune niche, and crosslink the immune‐evasion pathway to tumor oncogenic promotion.Methods:A 69‐year old female was admitted to the Cardiology Intensive Care Unit (ICU), for tachy‐/bradycardia. A cardiac computed tomography identified a voluminous rounded mass at the interatrial septum.The pathologic diagnosis was consistent with non‐GC DLBCL.Bone marrow (BM) biopsy detected CLL/SLL.Molecular analysis did not reveal SF3B1,NOTCH1 and TP53 alterations but evidenced the same IgVH mutation in both mass and BM specimens,proving a clonally‐related RS.Results:IHC stains for the cleaved NOTCH1‐intracellular domain (ICD) protein was negative on both RS lymphoma cells and the surrounding stroma, as in BM specimen. On the contrary, JAG1 was markedly positive both on large neoplastic lymphocytes and in the stromal compartment (Figure 1A). Conversely, JAG1 staining was moderate‐to‐weak on CLL lymphocytes in the BM, as well as in the stromal scaffold (Figure 1B), indicating differential expressions. Unexpectedly, JAG1 upregulation occurred independently of NOTCH1 receptor co‐activation. According to this, JAG1 may stimulate other pro‐survival cascades in the neoplastic B cells, transducing microenvironmental cues, coming from TME, where JAG1 itself was found highly expressed. In addition, we performed IHC stain for PD‐1, proving strong positivity on tumor‐infiltrating lymphocytes (TILs) (Figure 1C); moreover, PD‐L1 marked both neoplastic cells and stromal meshwork in the cardiac TME (Figure 1D).Summary/Conclusion:Our report proved the importance of IHC in characterizing JAG1‐NOTCH1 ligand‐receptor interactions in the RS pathogenesis, in order to anatomically characterize cell‐extrinsic pathways in vivo. We traced JAG1 upregulation, independently of NOTCH1 signaling activation and described PD‐1/PD‐L1 axis functional switching in the context of a very unique extra‐nodal RS localization, thus identifying novel potential actionable therapeutic targets.image

MicroRNA-34a inhibits epithelial-mesenchymal transition of lens epithelial cells by targeting Notch1

Posterior capsule opacification (PCO) is a common long-term complication of modern cataract surgery. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a crucial process in the development of PCO. The purpose of this study is to investigate the role of microRNA-34a (miR-34a) in the regulation of EMT and its target gene. Human LECs were treated with TGFβ2 to induce EMT as a model for PCO. The mRNA levels of miR-34a and EMT markers were examined by real-time quantitative polymerase chain reaction (qPCR). The expression level of miR-34a was downregulated, whereas that of Notch1 was upregulated in TGFβ2-induced EMT of LECs. Overexpression of miR-34a by transfection with miR-34a inhibited EMT of LECs and reduced the expression of Notch1; while, inhibition of miR-34a upregulated the expression of both Notch1 and its ligand Jagged1 in LECs. Luciferase reporter assays revealed that Notch1 gene was direct target of miR-34a. Moreover, DAPT, a specific inhibitor of Notch signaling pathway, reversed LEC-EMT. In addition, the expression level of miR-34a was downregulated, whereas that of Notch1 was upregulated in capsular opacification from cataract samples. MiR-34a can negatively regulate EMT of LECs by targeting Notch1. Therefore, miR-34a/Notch1 could serve as a potential therapeutic approach for the treatment of PCO.