Expression Of Notch Receptors Research Articles

Mesenchymal stem cells activate Notch signaling to induce regulatory dendritic cells in LPS-induced acute lung injury

BackgroundMesenchymal stem cells (MSCs) have been shown to alleviate acute lung injury (ALI) and induce the production of regulatory dendritic cells (DCregs), but the potential link between these two cell types remains unclear. The goal of this study was to investigate the effect and mechanism of MSC-induced regulatory dendritic cells in ALI mice.Material/methodsIn vivo experiments, C57BL/6 wild-type male mice were sacrificed at different times after intratracheal injection of LPS to observe changes in lung DC maturation and pathological damage. MSCs, DCregs or/and carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled DCs were administered to the mice by tail vein, and flow cytometry was performed to measure the phenotype of lung DCs and T cells. Lung injury was estimated by the lung wet weight/body weight ratio and histopathological analysis. In vitro, Western blotting or flow cytometry was used to detect the expression of Notch ligand or receptor in MSCs or DCs after coculture or LPS stimulation. Finally, in vivo and in vitro, we used the Notch signaling inhibitor DAPT to verify the effect of the Notch pathway on MSC-induced DCregs and their pulmonary protection.ResultsWe showed significant accumulation and maturation of lung DCs 2 h after intratracheal injection of LPS, which were positively correlated with the lung pathological injury score. MSC treatment alleviated ALI lung injury, accompanied by a decrease in the number and maturity of classical DCs in the lungs. CFSE-labeled DCs migrated to the lungs of ALI mice more than those of the normal group, and the elimination of CFSE-labeled DCs in the blood was slower. MSCs inhibited the migration of CFSE-labeled DCs to the lung and promoted their elimination in the blood. DCregs, which are obtained by contact coculture of mDCs with MSCs, expressed reduced levels of MHCII, CD86, CD40 and increased levels of PD-L1, and had a reduced ability to stimulate lymphocyte proliferation and activation (expression of CD44 and CD69). mDCs expressing Notch2 significantly increased after coculture with MSCs or rhJagged1, and MSCs expressed more Jagged1 after LPS stimulation. After stimulation of mDCs with recombinant Jagged1, DCs with low expression of MHCII, CD86 and CD40 were also induced, and the effects of both rhJagged1 and MSCs on DCs were blocked by the Notch inhibitor DAPT. Intra-airway DAPT reversed the inhibitory effect of mesenchymal stem cells on DC recruitment to the lungs and its maturation.ConclusionsOur results suggested that the recruitment and maturation of lung DCs is an important process in early ALI, MSCs attenuate LPS-induced ALI by inducing the production of DCregs by activating Notch signaling.

Abstract B23: PMN-MDSCs enhance CTC metastatic properties through reciprocal interactions via ROS/Notch/Nodal Nodal signaling

Abstract Intratumoral infiltration of myeloid-derived suppressor cells (MDSCs) is known to promote neoplastic growth by inhibiting the tumoricidal activity of T cells. However, direct interactions between patient-derived MDSCs and circulating tumors cells (CTCs) within the microenvironment of blood remain unexplored. Dissecting interplays between CTCs and circulatory MDSCs by heterotypic CTC/MDSC clustering is critical as a key mechanism to promote CTC survival and sustain the metastatic process. We characterized CTCs and polymorphonuclear-MDSCs (PMN-MDSCs) isolated in parallel from peripheral blood of metastatic melanoma and breast cancer patients by multiparametric flow cytometry. Transplantation of both cell populations in the systemic circulation of mice revealed significantly enhanced dissemination and metastasis in mice coinjected with CTCs and PMN-MDSCs compared to mice injected with CTCs or MDSCs alone. Notably, CTC/PMN-MDSC clusters were detected in vitro and in vivo either in patients’ blood or by longitudinal monitoring of blood from animals. This was coupled with in vitro coculturing of cell populations, demonstrating that CTCs formed physical clusters with PMN-MDSCs, and induced their protumorigenic differentiation through paracrine Nodal signaling, augmenting the production of reactive oxygen species (ROS) by PMN-MDSCs. These findings were validated by detecting significantly higher Nodal and ROS levels in blood of cancer patients in the presence of naïve, heterotypic CTC/PMN-MDSC clusters. Augmented PMN-MDSC ROS upregulated Notch1 receptor expression in CTCs through the ROS-NRF2-ARE axis, thus priming CTCs to respond to ligand-mediated (Jagged1) Notch activation. Jagged1-expressing PMN-MDSCs contributed to enhanced Notch activation in CTCs by engagement of Notch1 receptor. The reciprocity of CTC/PMN-MDSC bidirectional paracrine interactions and signaling was functionally validated in inhibitor-based analyses, demonstrating that combined Nodal and ROS inhibition abrogated CTC/PMN-MDSC interactions and led to a reduction of CTC survival and proliferation. This study provides seminal evidence showing that PMN-MDSCs, additive to their immunosuppressive roles, directly interact with CTCs and promote their dissemination and metastatic potency. Targeting CTC/PMN-MDSC heterotypic clusters and associated crosstalks can therefore represent a novel therapeutic avenue for limiting hematogenous spread of metastatic disease. Citation Format: Marc Sprouse, Thomas Welte, Debasish Boral, Monika Vishnoi, Haowen Liu, Isabella Glitza-Oliva, Dario Marchetti. PMN-MDSCs enhance CTC metastatic properties through reciprocal interactions via ROS/Notch/Nodal Nodal signaling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B23.

Notch Regulates Fibrocartilage Stem Cell Fate and Is Upregulated in Inflammatory TMJ Arthritis

The Notch pathway is critical for the development of the extracellular matrix in cartilage by regulating both anabolic and catabolic cellular activities. Similarly, Notch signaling plays a biphasic role in adult cartilage health and osteoarthritis by maintaining homeostasis and contributing to degeneration, respectively. The temporomandibular joint (TMJ) is the synovial joint of the craniofacial complex and is subject to injury and osteoarthritis. While Notch has been studied in axial skeletal joints, little is known about the role of Notch in TMJ development and disease. We identified fibrocartilage stem cells (FCSCs) localized within the TMJ condyle superficial zone niche that regenerate cartilage and repair joint injury. Here we investigate the role of Notch in regulating TMJ development and FCSC fate. Using a Notch reporter mouse, we discovered FCSCs localized within the TMJ superficial niche exhibit Notch activity during TMJ morphogenesis. We further showed that constitutively activating Notch promotes FCSC differentiation toward both cartilage and bone lineages, but inhibits adipogenesis. Using a TNF-α–induced TMJ inflammatory arthritis mouse model, we found that the expression of Notch receptors and ligands are upregulated and coupled with cells undergoing cartilage to bone transdifferentiation, which may contribute to TMJ pathogenesis. We also discovered that global Notch inhibition reduces osteogenic and chondrogenic differentiation of FCSCs. Together, these findings suggest that Notch is critical for FCSC fate specification and TMJ homeostasis, and reveal that inhibition of the Notch pathway may be a new therapeutic target for treating TMJ osteoarthritis.

Frequent Activation of Notch Signaling Pathway in Colorectal Cancers and Its Implication in Patient Survival Outcome.

Colorectal cancer is a major health concern as it ranks third in incidence and second major cause of cancer-related deaths worldwide. A leading cause of treatment failure has been attributed to cancer stem cells that can invariably resist existing chemotherapeutic regimens. Notch signaling pathway has been involved in the maintenance of stem cells besides being crucial in cell fate decision and embryonic development. This pathway has also been implicated in several human malignancies including colorectal cancer. We investigated mRNA expression of four Notch receptors (Notch1–4), five ligands (Jag1, Jag2, Dll1, Dll3, and Dll4), and four target genes (Hes1, Hes5, Hey1, and Hey2) using highly specific TaqMan gene expression assays in colorectal adenomas and cancers. Upregulated expression of Notch receptors ranged between 29 and 73% in colorectal cancers and between 11 and 56% in adenomas. Expression of Notch3 and Notch4 receptors was significantly higher in colorectal cancers compared to normal and adenoma tissues. The Jagged and Delta-like ligands were overexpressed between 25 and 52% in colorectal cancers, while in adenomas, it ranged between 0 and 33%. Combining the data for upregulation of receptors and ligands suggests that 86% colorectal cancers and 56% adenomas exhibited overexpression of Notch pathway genes in our cohort. Notch target genes were upregulated between 24 and 33% in colorectal cancers and between 11 and 22% in adenomas. Collating upregulation of Notch receptors and ligands with the target genes showed concordance in 58% colorectal tumors. Additionally, we evaluated expression of Notch receptors, ligands, and target genes with prognosis using the TCGA mRNA expression dataset. Patients overexpressing Notch3, Notch4, and Hey1 had significantly poorer overall survival relative to those having lower levels of these genes. Taken together, Notch signaling components are aberrantly overexpressed in colorectal tumors, and development of therapeutics targeting the Notch pathway may prove to be beneficial in the management of colorectal cancers.

Penicilazaphilone C, a New Azaphilone, Induces Apoptosis in Gastric Cancer by Blocking the Notch Signaling Pathway.

Penicilazaphilone C (PAC) is a novel azaphilonidal derivative isolated by our group that demonstrates good anticancer activities. Considering that its molecular mechanisms remain largely unknown, here we explore the molecular mechanisms of the anticancer activities of PAC against gastric cancer. The in vitro effects of PAC on cell growth, proliferation, and apoptosis were evaluated by MTT, BrdU, MTS, colony formation assays, Hoechst 33258 staining, and flow cytometry. Related proteins were examined by western blotting. Notch receptor expression was analyzed by RT-PCR. In vivo antitumor activities of PAC were observed in a nude mouse model. We found that compared to the controls, PAC treatment suppressed cell proliferation and promoted apoptosis in MGC-803 and SGC-7901 cells, and the Notch/PTEN/AKT axis was involved in the activating PAC-induced apoptosis. PAC treatment led to decreased levels of Notch (NTM), NICD, pPTEN, and pAKT compared to controls. PAC-induced inhibition of Notch-related protein expression levels and the resulting apoptosis were reversed by overexpression of Notch1 (NTM) or/and Notch2 (NTM). Moreover, PAC treatment clearly inhibited tumor growth in mice both bearing tumors derived from both MGC-803 and SGC-7901 cells. This work reveals that PAC induces the apoptosis by suppressing activation of Notch receptor proteolytic cleavage and subsequently blocking the PTEN/AKT signaling axis in gastric cancer cells. Thus, PAC is a potential alternative agent for the treatment of gastric cancer.

Notch1/2/3/4 are prognostic biomarker and correlated with immune infiltrates in gastric cancer.

Notch refers to a set of genes encoding a family of transmembrane receptors in mammalian cells. Previous evidence indicated that Notch receptors were implicated in the onset of gastric cancer. However, there is little evidence on the different genetic expression patterns of the four Notch receptors and their values for patient prognosis. Most recently, we investigated the transcriptional data of Notch receptors and related patient survival in patients with GC based on several databases, including ONCOMINE, GEPIA, Kaplan–Meier Plotter, cBioPortal and TIMER. According to our findings, gastric cancer tissues, compared with adjacent normal tissues presented a higher level of expression of Notch1/2/3. We also performed a survival analysis and noted that gastric cancer patients with high transcription levels of Notch1/2/3/4 had a low relapse-free survival. In gastric cancer patients, higher levels of infiltration in their CD4+ T cells, macrophages, neutrophils, and dendritic cells were positive associated with the expression of Notch receptors. Notch expression had significant association with diverse immune marker sets in gastric cancer. Overall, this study provides evidence that Notch1/2/3/4 could become the potential targets for precision treatment and new biomarkers in the prognosis of gastric cancer.

Notch receptor expression in Trypanosoma cruzi-infected human umbilical vein endothelial cells treated with benznidazole or simvastatin revealed by microarray analysis.

Chagas disease is a vector-borne disease caused by the protozoan parasite Trypanosoma cruzi. Current therapy involves benznidazole. Benznidazole and other drugs can modify gene expression patterns, improving the response to the inflammatory influx induced by T. cruzi and decreasing the endothelial activation or immune cell recruitment, among other effects. Here, we performed a microarray analysis of human umbilical vein endothelial cells (HUVECs) treated with benznidazole and the anti-inflammatory drugs acetylsalicylic acid or simvastatin and infected with T. cruzi. Parasitic infection produces differential expression of a set of genes in HUVECs treated with benznidazole alone or a combination with simvastatin or acetylsalicylic acid. The differentially expressed genes were involved in inflammation, adhesion, cardiac function, and remodeling. Notch1 and high mobility group B1 were genes of interest in this analysis due to their importance in placental development, cardiac development, and inflammation. Quantitative polymerase chain reaction confirmation of these two genes indicated that both are upregulated in the presence of benznidazole.

Role of HSP27 in the multidrug sensitivity and resistance of colon cancer cells.

Multidrug resistance in cancer cells is a primary factor affecting therapeutic efficacy. Heat shock 27 kD protein 1 (HSP27) is associated with cell apoptosis and resistance to chemotherapy. However, the mechanisms underlying HSP27-associated pathways in colon cancer cells remain unclear. Therefore, the present study used short hairpin (sh) RNA to inhibit HSP27 expression in colon cancer cells in order to investigate the effects in vitro and in vivo. Flow cytometry was used to investigate cell apoptosis and a xenograft model was employed to examine the tumorigenesis. Protein expression was measured by Western blotting. The results revealed that suppression of HSP27 expression significantly increased cell apoptosis, inhibited tumor growth and enhanced sensitivity to the anti-cancer agents 5-fluorouracil (5-FU) and vincristine (VCR). shHSP27 significantly decreased the expression of notch receptor 1 and the phosphorylation level of Akt and mTOR, and enhanced the effect of 5-FU and VCR. In conclusion, HSP27 suppression enhanced the sensitivity of colon cancer cells to 5-FU and VCR, and increased colon cancer cell apoptosis with and without chemotherapy. Therefore, the development of novel therapeutic agents that inhibit the expression of HSP27 may offer a new treatment option for colon cancer.

Indoxyl Sulfate-induced Vascular Calcification is mediated through Altered Notch Signaling Pathway in Vascular Smooth Muscle Cells.

Introduction: The aim of this study was to determine the role of Notch in indoxyl sulfate (IS)-induced vascular calcification (VC).Materials and methods: VC and expression of Notch-related and osteogenic molecules were examined in Dahl salt-sensitive (DS), DS hypertensive (DH), and DH IS-treated rats (DH+IS). The effects of IS on expression of Notch receptors, apoptotic activity, and calcification were examined in cultured aortic smooth muscle cells (SMCs).Results: Medial calcification was noted only in aortas and coronary arteries of DH+IS rats. Notch1, Notch3, and Hes-1 were expressed in aortic SMCs of all rats, but only weakly in the central areas of the media and around the calcified lesions in DH+IS rats. RT-PCR and western blotting of DH+IS rat aortas showed downregulation of Notch ligands, Notch1 and Notch3, downstream transcriptional factors, and SM22, and conversely, overexpression of osteogenic markers. Expression of Notch1 and Notch3 in aortic SMCs was highest in incubation under 500 μM IS for 24hrs, and then decreased time- and dose-dependently. Coupled with this decrease, IS increased caspase 3/7 activity and TUNEL-positive aortic SMCs. In addition, pharmacological Notch signal inhibition with DAPT induced apoptosis in aortic SMCs. ZVAD, a caspase inhibitor abrogated IS-induced and DAPT-induced in vitro vascular calcification. Knockdown of Notch1 and Notch3 cooperatively increased expression of osteogenic transcriptional factors and decreased expression of SM22.Conclusion: Our results suggested that IS-induced VC is mediated through suppression of Notch activity in aortic SMCs, induction of osteogenic differentiation and apoptosis.

The Use of Single-Cell Transcriptomics’ to Detect Cellular Transition through Notch Signaling and Receptor Expression in Differentiated Kidney Collecting Tubule Cells in Adult Mice: A Review

The Use of Single-Cell Transcriptomics’ to Detect Cellular Transition through Notch Signaling and Receptor Expression in Differentiated Kidney Collecting Tubule Cells in Adult Mice: A Review

LncRNA Rmst acts as an important mediator of BMP9-induced osteogenic differentiation of mesenchymal stem cells (MSCs) by antagonizing Notch-targeting microRNAs.

Understanding the bone and musculoskeletal system is essential to maintain the health and quality of life of our aging society. Mesenchymal stem cells (MSCs) can undergo self-renewal and differentiate into multiple tissue types including bone. We demonstrated that BMP9 is the most potent osteogenic factors although molecular mechanism underlying BMP9 action is not fully understood. Long noncoding RNAs (lncRNAs) play important regulatory roles in many physiological and/or pathologic processes. Here, we investigated the role of lncRNA Rmst in BMP9-induced osteogenic differentiation of MSCs. We found that Rmst was induced by BMP9 through Smad signaling in MSCs. Rmst knockdown diminished BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro, and attenuated BMP9-induced ectopic bone formation. Silencing Rmst decreased the expression of Notch receptors and ligands. Bioinformatic analysis predicted Rmst could directly bind to eight Notch-targeting miRNAs, six of which were downregulated by BMP9. Silencing Rmst restored the expression of four microRNAs (miRNAs). Furthermore, an activating Notch mutant NICD1 effectively rescued the decreased ALP activity caused by Rmst silencing. Collectively, our results strongly suggest that the Rmst-miRNA-Notch regulatory axis may play an important role in mediating BMP9-induced osteogenic differentiation of MSCs.

Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade.

Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase Mfng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.

MS05.03 Notch Signalling

The Notch signaling pathway is a highly conserved pathway that has a vital role in embryonic development and post-embryonic functions such as hematopoiesis, neural cell development and angiogenesis (1). The signaling pathway is activated by the interaction of 4 Notch receptors and their interactions with 5 different ligands. Signaling is characterized by juxtacrine interaction between ligands and receptors on neighboring cells or within the same cell. Alterations in this pathway have been detected in several tumors both as tumor promoter and tumor suppressor. Notch signaling does appear to have relevance in non-small cell lung cancers (NSCLC) (2). In approximately 30% of NSCLCs loss of Numb, a negative regulator of Notch1, leads to increased Notch activity and about 10% of NSCLCs have gain of function mutations in Notch1. A meta-analysis demonstrated that higher expression of Notch1 was correlated with more advanced tumors (3). In addition, higher expressions of both Notch1 and Notch3 were associated with poor prognosis (4,5). In pre-clinical studies inhibition of Notch3 signaling has reduced growth of lung cancers. In addition, it has been demonstrated that there is cross talk between Notch3 and EGFR pathways and inhibition of both pathways can induce expression of anti-apoptotic protein BIM. Finally, Notch signaling may have a role in induction of the epithelial to mesenchymal phenotype (EMT) in cancers (6). EMT is known to be associated with resistance to both cytotoxic agents and targeted agents and inhibition of Notch signaling can not only reverse EMT but also can enhance the anti-tumor activity of cytotoxic agents such as cisplatin and targeted agents such as erlotinib. The EMT phenotype is frequently observed in a sub-population of cancer cells with self-renewal capacity or cancer stem cells. Notch signaling may be crucial to survival of cancer stem cells and persistence of this population may contribute to resistance to therapeutic agents. In squamous cell lung cancers Notch signaling may have tumor suppressive properties (7). Loss of function mutations in Notch family of genes, predominantly in Notch receptors, are frequently identified in several squamous cell cancers including squamous cell cancer of the lung. Similarly, loss of function mutations in Notch genes, particularly Notch1 have been identified in small cell lung cancer (SCLC). Expression of Notch receptor in a mouse model of SCLC reduced tumor burden, suggesting its tumor suppressive properties. The expression of DLL3, one of the Notch ligands is induced in SCLC by a key transcription factor ASCL1.DLL3 is shown to downregulate Notch signaling in SCLCs and enhance the carcinogenic phenotype. All the above data suggest that Notch signaling is highly contextual. In some tumors this pathway may have tumor suppressive properties but in others tumor promoting properties. Defining the role of this pathway in tumor types may guide development of therapeutic strategies targeting the Notch signaling pathway. Bigas A, Espinosa L. The multiple usages of Notch signaling in development, cell differentiation and cancer. Curr Opin Cell Biol 2018;55:1-7. Westhoff B, Colaluca IN, D’Ario G, et al. Alterations of the Notch pathway in lung cancer. Proc Natl Acad Sci 2009;106:22293-8. Yuan X, Wu H, Xu H, et al. Meta-analysis reveals the correlation of Notch signaling with non-small cell lung cancer progression and prognosis. Sci Rep 2015;5:10338. Donnem T, Andersen S, Al-shibili K, et al. Prognostic impact of Notch ligands and receptors in nonsmall cell lung cancer: coexpression of Notch-1 and vascular endothelial growth factor-A predicts poor survival. Cancer 2010:116:5674-85. Hassan WA, Yoshida R, Kudoh S, et al. Evaluation of role of Notch3 signaling pathway in human lung cancer cells. J Cancer Res Clin Oncol 2016;142:981-93. Yuan X, Wu H, Han N, et al. Notch signaling and EMT in non-small cell lung cancer: biological significance and therapeutic application. J Hematol Oncol 2014;7:87. Nowell CS, Radtke F. Notch as a tumor suppressor. Nat Rev Cancer 2017;17:145-159. squamous, Tumor suppressor, NOTCH

FLI-06 Intercepts Notch Signaling And Suppresses The Proliferation And Self-renewal Of Tongue Cancer Cells.

PurposeThe Notch signaling pathway plays an oncogenic role in tongue squamous cell carcinoma. The aim of this study was to inhibit the proliferation and self-renewal of tongue cancer cells by applying Notch signaling pathway inhibitor FLI-06 (Selleck, USA) and to lay a foundation for the clinically targeted treatment of tongue cancer for the future.MethodsThe mRNA expression level of Notch1 and the overall survival rate of patients with tongue cancer were examined by analyzing the TCGA database. Tongue cancer cells were treated with FLI-06. Cell proliferation, apoptosis, and stem cell self-renewal ability were tested in appropriate ways. A xenograft mouse model was established to observe tumor growth.ResultsFrom the TCGA data, we demonstrated that patients with high expression of Notch1 had a poor prognosis. We observed that the Notch signaling pathway inhibitor FLI-06 can restrain the activation of the Notch signaling pathway, decrease cell proliferation and induce cell apoptosis in vitro. The xenograft experiment indicated that intraperitoneal injection of FLI-06 inhibited tumor growth and increased cell apoptosis. FLI-06 suppressed both the mRNA and protein expression of Notch receptor and Notch targeted genes. We also observed that FLI-06 suppressed the proliferation of tongue cancer stem cells.ConclusionFLI-06 can block the proliferation and self-renewal of tongue cancer cells. It is inferred that this compound, which inhibits the Notch signaling pathway, may serve as a potential targeted drug for the treatment of tongue cancer in the clinic.

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.

Misoprostol-Induced Modification of the Notch Signaling Pathway in the Human Cervix.

The complex and multifactorial mechanisms that initiate and sustain the early labor process in the human uterus and cervix are still not well defined. Cervical maturation or ripening is likely to play a key role in preparing for birth. Prostaglandins have many different functions, including the regulation of uterine contractility and structure during pregnancy. The prostaglandin E1 analogue misoprostol is frequently used as a uterotonic and cervical ripening agent. Notch is a transmembrane receptor family responsible for basic functions such as cell survival, cell-cell communication, and differentiation and decidualization in pregnancy. However, our understanding of the effect of Notch signaling on the cervical ripening process is limited. This study was conducted in 20 pregnant women aged at 12 to 20 weeks of gestation undergoing medical abortion for fetal or maternal indications. True-Cut needle biopsies were taken from the anterior cervix 4 hours after oral ingestion of 200-μg misoprostol or before the ingestion of misoprostol in the control group. Cervical expression of Notch receptors and ligands changed during the early phase of prostaglandin-induced preterm labor. Four hours after the administration of misoprostol, it was seen that N1 expression increased in muscle, while DLL1 and J2 expression increased in blood vessels, and N4 expression increased in macrophages. Knowing the mechanisms that initiate preterm birth is the most important step in planning the treatments and actions to prevent premature birth. As a signal that affects and perhaps directs preterm labor, Notch is prone to be an important actor in this process.

Abstract 1291: Notch2 inhibition as a therapeutic intervention in osteosarcoma

Abstract Notch signaling pathway is a mediator of cell differentiation and is critical for normal bone development. Four functional Notch ligands DLL1 and DLL4, and JAG1 and JAG2 showed various levels of affinity for Notch1-4 receptors. Dose dependent Notch signaling activation or blocking is attributed to anchored or soluble form of Notch ligands binding. Interestingly, Notch pathway is shown to play a dual role, either oncogenic or tumor suppressive, depending on signal dose and tissue-context. To understand Notch signaling activities in osteosarcoma (OS), we first studied the cell surface levels of Notch receptor expression in OS cell lines and patient derived xenograft (pdx) models. Cytometry based screening of NOTCH1-4 receptors showed NOTCH2 as a predominant surface expressed protein in most of the samples. Interestingly, the canonical Notch target genes HES1 and DTX1 were higher (~5-fold) in OS cells compared to mesenchymal cells (MSC). Subsequently, we evaluated selected Notch pathway gene expression in 48 patients with recurrent/metastatic OS by RNAseq analysis. High expression of Notch pathway-related genes was seen in a subset of patients. NOTCH2 expression was comparable between primary and metastatic OS specimens, highest as compared to other Notch receptors and was significantly higher than normal tissue. Thus we aimed to elucidate soluble Notch ligands mediated blocking of Notch signaling using immunoglobulin-G bound DLL-1, -3, and -4 and JAG-1, and -2 in in vitro studies using OS cells. Soluble DLL1-Fc treatment increased OS cell death as compared to the other Notch ligands as well as control-Fc cells. Addition of soluble DLL1 resulted in the decreased expression of Notch downstream genes such as HES1, DTX1 and HEY1. Taken together, these findings identify soluble DLL1 as a potent Notch pathway inhibitor in OS in vitro and support further exploration of NOTCH2 as a potential therapeutic target in OS. Citation Format: Sankaranarayanan Kannan, John A. Livingston, Michael Roth, Jonathan Benjamin, Yifei Wang, Zhongting Zhang, Wendong Zhang, Chia-Chin Wu, Hannah Beird, Andrew Futreal, Richard Gorlick. Notch2 inhibition as a therapeutic intervention in osteosarcoma [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 1291.

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.

Sa1376 – Expression of Notch Receptors and Their Role As Differentiating Markers in Pancreatic Ductal Adenocarcinoma

Sa1376 – Expression of Notch Receptors and Their Role As Differentiating Markers in Pancreatic Ductal Adenocarcinoma

PMN-MDSCs Enhance CTC Metastatic Properties through Reciprocal Interactions via ROS/Notch/Nodal Signaling.

Intratumoral infiltration of myeloid-derived suppressor cells (MDSCs) is known to promote neoplastic growth by inhibiting the tumoricidal activity of T cells. However, direct interactions between patient-derived MDSCs and circulating tumors cells (CTCs) within the microenvironment of blood remain unexplored. Dissecting interplays between CTCs and circulatory MDSCs by heterotypic CTC/MDSC clustering is critical as a key mechanism to promote CTC survival and sustain the metastatic process. We characterized CTCs and polymorphonuclear-MDSCs (PMN-MDSCs) isolated in parallel from peripheral blood of metastatic melanoma and breast cancer patients by multi-parametric flow cytometry. Transplantation of both cell populations in the systemic circulation of mice revealed significantly enhanced dissemination and metastasis in mice co-injected with CTCs and PMN-MDSCs compared to mice injected with CTCs or MDSCs alone. Notably, CTC/PMN-MDSC clusters were detected in vitro and in vivo either in patients’ blood or by longitudinal monitoring of blood from animals. This was coupled with in vitro co-culturing of cell populations, demonstrating that CTCs formed physical clusters with PMN-MDSCs; and induced their pro-tumorigenic differentiation through paracrine Nodal signaling, augmenting the production of reactive oxygen species (ROS) by PMN-MDSCs. These findings were validated by detecting significantly higher Nodal and ROS levels in blood of cancer patients in the presence of naïve, heterotypic CTC/PMN-MDSC clusters. Augmented PMN-MDSC ROS upregulated Notch1 receptor expression in CTCs through the ROS-NRF2-ARE axis, thus priming CTCs to respond to ligand-mediated (Jagged1) Notch activation. Jagged1-expressing PMN-MDSCs contributed to enhanced Notch activation in CTCs by engagement of Notch1 receptor. The reciprocity of CTC/PMN-MDSC bi-directional paracrine interactions and signaling was functionally validated in inhibitor-based analyses, demonstrating that combined Nodal and ROS inhibition abrogated CTC/PMN-MDSC interactions and led to a reduction of CTC survival and proliferation. This study provides seminal evidence showing that PMN-MDSCs, additive to their immuno-suppressive roles, directly interact with CTCs and promote their dissemination and metastatic potency. Targeting CTC/PMN-MDSC heterotypic clusters and associated crosstalks can therefore represent a novel therapeutic avenue for limiting hematogenous spread of metastatic disease.