NOTCH1 signaling induces pathological vascular permeability in diabetic retinopathy.

Diabetic macular edema is a major complication of diabetes resulting in loss of central vision. Although heightened vessel leakiness has been linked to glial and neuronal-derived factors, relatively little is known on the mechanisms by which mature endothelial cells exit from a quiescent state and compromise barrier function. Here we report that endothelial NOTCH1 signaling in mature diabetic retinas contributes to increased vascular permeability. By providing both human and mouse data, we show that NOTCH1 ligands JAGGED1 and DELTA LIKE-4 are up-regulated secondary to hyperglycemia and activate both canonical and rapid noncanonical NOTCH1 pathways that ultimately disrupt endothelial adherens junctions in diabetic retinas by causing dissociation of vascular endothelial-cadherin from ß-catenin. We further demonstrate that neutralization of NOTCH1 ligands prevents diabetes-induced retinal edema. Collectively, these results identify a fundamental process in diabetes-mediated vascular permeability and provide translational rational for targeting the NOTCH pathway (primarily JAGGED1) in conditions characterized by compromised vascular barrier function.

Therapies targeting Frizzled-7/ß-catenin pathway prevent the development of pathological angiogenesis in an ischemic retinopathy model.

Retinopathies remain major causes of visual impairment in diabetic patients and premature infants. Introduction of anti-angiogenic drugs targeting vascular endothelial growth factor (VEGF) has transformed therapy for these proliferative retinopathies. However, limitations associated with anti-VEGF medications require to unravel new pathways of vessel growth to identify potential drug targets. Here, we investigated the role of Wnt/Frizzled-7 (Fzd7) pathway in a mouse model of oxygen-induced retinopathy (OIR). Using transgenic mice, which enabled endothelium-specific and time-specific Fzd7 deletion, we demonstrated that Fzd7 controls both vaso-obliteration and neovascular phases (NV). Deletion of Fzd7 at P12, after the ischemic phase of OIR, prevented formation of aberrant neovessels into the vitreous by suppressing proliferation of endothelial cells (EC) in tufts. Next we validated in vitro two Frd7 blocking strategies: a monoclonal antibody (mAbFzd7) against Fzd7 and a soluble Fzd7 receptor (CRD). In vivo a single intravitreal microinjection of mAbFzd7 or CRD significantly attenuated retinal neovascularization (NV) in mice with OIR. Molecular analysis revealed that Fzd7 may act through the activation of Wnt/ß-catenin and Jagged1 expression to control EC proliferation in extra-retinal neovessels. We identified Fzd7/ß-catenin signaling as new regulator of pathological retinal NV. Fzd7 appears to be a potent pharmacological target to prevent or treat aberrant angiogenesis of ischemic retinopathies.

MicroRNA-489-3p Represses Hepatic Stellate Cells Activation by Negatively Regulating the JAG1/Notch3 Signaling Pathway.

BACKGROUND: The transformation of hepatic stellate cells (HSCs) into collagen-producing myofibroblasts is a key event in hepatic fibrogenesis. Recent studies have shown that microRNAs (miRNAs) play a critical role in the transformation of HSCs. However, the function of miR-489-3p in liver fibrosis remains unclear. METHODS: Here, we detected the levels of miR-489-3p and jagged canonical Notch ligand 1 (JAG1) in liver fibrosis by using CCl4-treated rats as an in vivo model and transforming growth factor-beta 1 (TGF-ß1)-treated HSC cell lines LX-2 and HSC-T6 as in vitro models. The expression of profibrotic markers was affected by transfecting LX-2 cells with either miR-489-3p mimic or si-JAG1. A dual-luciferase reporter assay was carried out to study the interaction of JAG1 with miR-489-3p. RESULTS: We found that miR-489-3p was remarkably decreased while JAG1 was increased in liver fibrosis models both in vivo and in vitro. Overexpression of miR-489-3p reduced the expression of profibrotic markers and the activation of LX-2 cells induced by TGF-ß1. Moreover, miR-489-3p decreased the expression of jagged canonical Notch ligand 1 (JAG1) in LX-2 cells by interacting with its 3'-UTR. As JAG1 is a Notch ligand, decreased JAG1 by miR-489-3p inhibited the Notch signaling pathway. Moreover, the downregulation of JAG1 inhibited the expression of fibrotic markers. CONCLUSION: Our results indicate that miR-489-3p can inhibit HSC activation by inhibiting the JAG1/Notch3 signaling pathway.

Mechanosensitivity of Jagged-Notch signaling can induce a switch-type behavior in vascular homeostasis.

Hemodynamic forces and Notch signaling are both known as key regulators of arterial remodeling and homeostasis. However, how these two factors integrate in vascular morphogenesis and homeostasis is unclear. Here, we combined experiments and modeling to evaluate the impact of the integration of mechanics and Notch signaling on vascular homeostasis. Vascular smooth muscle cells (VSMCs) were cyclically stretched on flexible membranes, as quantified via video tracking, demonstrating that the expression of Jagged1, Notch3, and target genes was down-regulated with strain. The data were incorporated in a computational framework of Notch signaling in the vascular wall, where the mechanical load was defined by the vascular geometry and blood pressure. Upon increasing wall thickness, the model predicted a switch-type behavior of the Notch signaling state with a steep transition of synthetic toward contractile VSMCs at a certain transition thickness. These thicknesses varied per investigated arterial location and were in good agreement with human anatomical data, thereby suggesting that the Notch response to hemodynamics plays an important role in the establishment of vascular homeostasis.

Olfactory Sensory Neurons Control Dendritic Complexity of Mitral Cells via Notch Signaling.

Mitral cells (MCs) of the mammalian olfactory bulb have a single primary dendrite extending into a single glomerulus, where they receive odor information from olfactory sensory neurons (OSNs). Molecular mechanisms for controlling dendritic arbors of MCs, which dynamically change during development, are largely unknown. Here we found that MCs displayed more complex dendritic morphologies in mouse mutants of Maml1, a crucial gene in Notch signaling. Similar phenotypes were observed by conditionally misexpressing a dominant negative form of MAML1 (dnMAML1) in MCs after their migration. Conversely, conditional misexpression of a constitutively active form of Notch reduced their dendritic complexity. Furthermore, the intracellular domain of Notch1 (NICD1) was localized to nuclei of MCs. These findings suggest that Notch signaling at embryonic stages is involved in the dendritic complexity of MCs. After the embryonic misexpression of dnMAML1, many MCs aberrantly extended dendrites to more than one glomerulus at postnatal stages, suggesting that Notch signaling is essential for proper formation of olfactory circuits. Moreover, dendrites in cultured MCs were shortened by Jag1-expressing cells. Finally, blocking the activity of Notch ligands in OSNs led to an increase in dendritic complexity as well as a decrease in NICD1 signals in MCs. These results demonstrate that the dendritic complexity of MCs is controlled by their presynaptic partners, OSNs.

Gremlin activates the Notch pathway linked to renal inflammation.

Preclinical studies suggest that Gremlin participates in renal damage and could be a potential therapeutic target for human chronic kidney diseases. Inflammation is a common characteristic of progressive renal disease, and therefore novel anti-inflammatory therapeutic targets should be investigated. The Notch signaling pathway is involved in kidney development and is activated in human chronic kidney disease, but whether Gremlin regulates the Notch pathway has not been investigated. In cultured tubular cells, Gremlin up-regulated gene expression of several Notch pathway components, increased the production of the canonical ligand Jagged-1, and caused the nuclear translocation of active Notch-1 (N1ICD). In vivo administration of Gremlin into murine kidneys elicited Jagged-1 production, increased N1ICD nuclear levels, and up-regulated the gene expression of the Notch effectors hes-1 and hey-1 All these data clearly demonstrate that Gremlin activates the Notch pathway in the kidney. Notch inhibition using the γ-secretase inhibitor DAPT impaired renal inflammatory cell infiltration and proinflammatory cytokines overexpression in Gremlin-injected mice and in experimental models of renal injury. Moreover, Notch inhibition blocked Gremlin-induced activation of the canonical and noncanonical nuclear factor-κB (NF-κB) pathway, identifying an important mechanism involved in the anti-inflammatory actions of Notch inhibition. In conclusion, Gremlin activates the Notch pathway in the kidney and this is linked to NF-κB-mediated inflammation, supporting the hypothesis that Notch inhibition could be a potential anti-inflammatory strategy for renal diseases.

Lineage tracing of Sox2-expressing progenitor cells in the mouse inner ear reveals a broad contribution to non-sensory tissues and insights into the origin of the organ of Corti.

The transcription factor Sox2 is both necessary and sufficient for the generation of sensory regions of the inner ear. It regulates expression of the Notch ligand Jag1 in prosensory progenitors, which signal to neighboring cells to up-regulate Sox2 and sustain prosensory identity. However, the expression pattern of Sox2 in the early inner ear is very broad, suggesting that Sox2-expressing progenitors form a wide variety of cell types in addition to generating the sensory regions of the ear. We used Sox2-CreER mice to follow the fates of Sox2-expressing cells at different stages in ear development. We find that Sox2-expressing cells in the early otocyst give rise to large numbers of non-sensory structures throughout the inner ear, and that Sox2 only becomes a truly prosensory marker at embryonic day (E)11.5. Our fate map reveals the organ of Corti derives from a central domain on the medial side of the otocyst and shows that a significant amount of the organ of Corti derives from a Sox2-negative population in this region.

Diabetes-Induced Jagged1 Overexpression in Endothelial Cells Causes Retinal Capillary Regression in a Murine Model of Diabetes Mellitus: Insights Into Diabetic Retinopathy.

BACKGROUND: Several mechanisms have been proposed to account for diabetes-induced microvasculopathy (DMV). Although Notch signaling was reported to be affected by glucose metabolism in endothelial cells during developmental angiogenesis, it has not been investigated in vascular remodeling of adult capillaries in relation to diabetes mellitus. METHODS: We induced diabetes mellitus in 8-week-old adult mice by intravenously administering streptozotocin. After 6 weeks, we harvested organs, including retina, heart, and skeletal muscle, and evaluated the capillaries with immunofluorescence and confocal microscopy. We modulated endothelial Notch signaling using chemical inhibitors in wild-type mice or transgenic mice, inducing conditional knockout of Jagged1 or Mib1. RESULTS: DMV was characterized by capillary remodeling, regression, and decreased density. Notch ligand Jagged1, but not δ-like ligand 4, was markedly increased in endothelial cells of diabetic mice. Using endothelium-specific Jagged1 knockdown mice, we found that blocking Jagged1 prevented DMV even under diabetic conditions. Furthermore, in the inducible endothelium-specific Jagged1 knockdown mice, blocking Jagged1 even at 4 weeks after the establishment of DMV could reverse it, leading to normalization of retinal vasculature. A search for downstream signals revealed that diabetes mellitus decreased the nuclear localization of Notch1 intracellular domain and reduced the expression of VE-cadherin and N-cadherin in endothelial cells. Chemical Notch inhibition phenocopied DMV in normal mice. CONCLUSIONS: Our findings indicate that diabetes mellitus induces Jagged1 overexpression and suppresses Notch signaling in endothelial cells, leading to DMV in adult mice. We conclude that dysregulated intercellular Notch signaling may be a novel mechanism of DMV.

miR-199a-3p inhibits cell proliferation and induces apoptosis by targeting YAP1, suppressing Jagged1-Notch signaling in human hepatocellular carcinoma.

BACKGROUND: miR-199a-3p was significantly downregulated in the majority of human hepatocellular carcinoma (HCC) tissues and HCC cell lines. Yes associated protein 1 (YAP1) was overexpressed in human HCC, which promoted HCC development and progression by upregulating Jagged1 and activating the Notch pathway. We searched potential targets of miR-199a-3p with DIANA, TargetScan and PicTar tools, and found that YAP1 is one of the potential targets. Based on these findings, we speculated that miR-199a-3p might suppress HCC growth by targeting YAP1, downregulating Jagged1 and suppressing the Notch pathway. RESULTS: We determined the expression of miR-199a-3p and YAP1 by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found downregulation of miR-199a-3p and upregulation of YAP1 in HCC cell lines. Cell proliferation and apoptosis assays showed that miR-199a-3p suppresses HCC cell proliferation and promotes apoptosis, and knockdown of YAP1 has similar role. Furthermore, we verified that miR-199a-3p can directly target YAP1. We further investigated and confirmed that miR-199a-3p and YAP1 regulate HCC cell proliferation and apoptosis through Jagged1-Notch signaling. CONCLUSION: miR-199a-3p targets YAP1, downregulates Jagged1 and suppresses the Notch signaling to inhibit HCC cell proliferation and promote apoptosis. These findings provide new insights into the mechanism by which miR-199a-3p suppresses HCC cell proliferation and induces apoptosis.

TGFß1-Smad3-Jagged1-Notch1-Slug signaling pathway takes part in tumorigenesis and progress of tongue squamous cell carcinoma.

BACKGROUND: TGFß1 and Smad3 play an important role in the process of EMT. TGFß1 regulates the expression of Jagged1 by modulating Notch signaling. Jagged1 is related to tumor invasion, metastasis, chemotherapy resistance, and tumor immune escape. The aims of this study are to examine deregulation of TGFß1-Smad3-Jagged1-Notch1-Slug signaling in TSCC and to investigate its roles in TSCC progression. MATERIALS AND METHODS: Notch1, Smad3, Jagged1 and Slug proteins and mRNA expression were detected in specimens from 89 cases of patients. We analyzed the correlation between their expressions and histological grade, clinical stage and lymph node metastasis. RESULTS: Notch1, Smad3, Jagged1 and Slug mRNA expressions in TSCC were higher than normal tissue (P <0.05). The protein expression of Notch1 and Smad3 in TSCC were higher (χ(2) =7.30, P <0.01 and χ(2) = 5.84, P <0.05). Notch1 and Smad3 expressions were correlated with clinical stage (χ(2) =18.81, P<0.01; χ(2) =22.29, P<0.01), but not Jagged1 (χ(2) =0.53, P>0.05). The Slug protein expression was correlated with clinical stage. The positive rate of Notch1 was higher in lymph node metastases positive cases (χ(2) =7.30, P<0.01). Moreover, higher expression of Jagged1 was found in lymph node positive cases (χ(2) =10.82, P<0.01). CONCLUSIONS: The key protein expression in TGFß1-Smad3-Jagged1-Notch1-Slug signaling pathway significantly correlated with the clinicopathological features of TSCC patients. It's potential as a biomarker and a novel therapeutic target for TSCC patients at risk of metastasis. It may play an irreplaceable role in TSCC progression which may attribute to promoting EMT which enhances cell migration, invasion and metastasis.

Identifying specific Notch1 target proteins in lung carcinoma cells.

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

Depletion of circ_0007841 inhibits multiple myeloma development and BTZ resistance via miR-129-5p/JAG1 axis.

Circular RNAs (circRNAs) possess important regulatory effects on multiple myeloma (MM) progression. Here, we aimed at exploring the function of circ_0007841 in MM and the underlying molecular mechanism. Expression of circ_0007841, microRNA (miR)-129-5p and Jagged1 (JAG1) was determined via qRT-PCR or western blot assay. Methyl thiazolyl tetrazolium (MTT) assay was applied to examine cell viability and IC50 value of MM cells to bortezomib (BTZ). Colony formation assay was performed to analyze cell proliferation. Moreover, cell apoptosis was assessed by flow cytometry and western blot analysis. Cell metastasis was evaluated by wound healing assay and Transwell assay. Function of circ_0007841 in vivo was determined by xenograft tumor assay. Target relationship between miR-129-5p and circ_0007841 or JAG1 was confirmed via dual-luciferase reporter, RNA immunoprecipitation (RIP) and pull-down assays. The up-regulation of circ_0007841 and JAG1, and the down-regulation of miR-129-5p were detected in MM bone marrow aspirates and cells. Circ_0007841 knockdown significantly repressed cell proliferation, chemoresistance, and metastasis, while contributed to apoptosis of MM cells in vitro, and reduced tumor growth in vivo. Circ_0007841 targeted miR-129-5p, and miR-129-5p inhibition reversed impact of silencing of circ_0007841 on MM cells. JAG1 was a mRNA target of miR-129-5p, whose overexpression could undermine the miR-129-5p-mediated effects on MM cells. Circ_0007841 positively regulated JAG1 expression via absorbing miR-129-5p. Circ_0007841 knockdown inhibited MM cell proliferation, metastasis and chemoresistance through modulating miR-129-5p/JAG1 axis, suggesting that circ_0007841 might serve as a potential therapeutic target of MM.

≤ Cyclin D1 protein affecting global women's health by regulating HPV mediated adenocarcinoma of the uterine cervix.

Adenocarcinoma (ADC) of the uterine cervix (UC) is a rare form of cervical cancer (CC) caused due to the infection of Human Papilloma Virus (HPV). Cyclin D1 is one of the downstream targets of aberrantly activated Notch signaling, contribute to the etiology of CC. However, little is known about the role of Cyclin D1 in the modulation of cervical ADC and is controversial. The purpose of this study is to determine the role of Cyclin D1 protein and to elucidate the combined analysis with Notch signaling proteins in HPV associated ADCs of CC. A total of 60 biopsy samples (40 normal and 20 ADCs of CC) were analyzed for the expression of Cyclin D1 in HPV associated ADCs via immunohistochemistry and by immunoblotting. HPV-16 positive ADC patients showed a strong association with the Cyclin D1 expression (p = 0.007). The significant mean difference (p = 0.0001) and the pairwise comparison between Cyclin D1/JAG1 (p = 0.0001), and Cyclin D1/Notch-3 (p = 0.0001) were observed. The above Notch signaling proteins showed their synergistic role in modulating Cyclin D1 which in-turn regulates HPV-16 associated ADC of the uterine cervix (UC), affecting women's global health.

miR-26b-5p suppresses proliferation and promotes apoptosis in multiple myeloma cells by targeting JAG1.

BACKGROUND: Though the levels of diagnosis and treatment of multiple myeloma (MM) have been largely improved recent years, the prognosis of these patients remain unacceptable. It is urgent for us to discover the exact mechanism and determine some new indicators for MM. MiRNAs play a critical role in the occurrence and progression of cancers, including MM. MiR-26b-5p has been reported to be closely related to cells proliferation in human pulmonary cancer, hepatocellular carcinoma and so on. MATERIAL AND METHODS: Here, we measured the expression of miR-26b-5p in MM samples and cell lines by real-time PCR. Then, Kaplan-Meier Curves were applied to assess the effect of miR-26b-5p expression on MM patients prognosis. Functionally, MTT assay and Flow cytometry were conducted to explore the functions of miR-26b-5p in cells proliferation and apoptosis. Furthermore, bioinformatics tools, Pearson's correlation coefficient analysis, gain-and loss of-function experiments and rescue experiment were used to determine the relationship between JAG1 and miR-26b-5p in MM cells. In addition, we also confirmed the role of JAG1 in MM cells proliferation and apoptosis by gain-and loss of-function experiments. RESULTS: Here, we reported for the first time that miR-26b-5p was under-expressed in MM by real-time PCR. Clinically, Kaplan-Meier Curves showed that MM patients with lower miR-26b-5p expression had worse prognosis. Functionally, MTT assay revealed that miR-26b-5p inhibited cells proliferation. Flow cytometry indicated that miR-26b-5p accelerated tumor cells apoptosis. Furthermore, bioinformatics tools, Pearson's correlation coefficient analysis gain-and loss of-function experiments showed that JAG1 was the target of miR-26b-5p in MM cells. And, gain-and loss of-function experiments for JAG1 confirmed that JAG1 was an oncogene in MM cells. What's more, rescue experiment showed that JAG1 mediated the function of miR-26b-5p in MM cells. CONCLUSION: MiR-26b-5p acts as a tumor suppressor through suppressing cells proliferation and inducing cells apoptosis via directly targeting JAG1 in MM. MiR-26b-5p could be a potential and ponderable tumor target for MM in future.

Studies on sporadic non-syndromic thoracic aortic aneurysms: 1. Deregulation of Jagged/Notch 1 homeostasis and selection of synthetic/secretor phenotype smooth muscle cells.

Background Sporadic non-syndromic thoracic aortic aneurysms (SNSTAAs) are less well understood than familial non-syndromic or syndromic ones. The study aimed at defining the peculiar morphologic and molecular changes occurring in the media layer of SNSTAAs. Design This study was based on a single centre design. Methods Media layer samples taken from seven carefully selected SNSTAAs and seven reference patients (controls) were investigated via quantitative polymerase chain reaction, proteomics-bioinformatics, immunoblotting, quantitative histology, and immunohistochemistry/immunofluorescence. Results In SNSTAAs media, aortic smooth muscle cells numbers were halved due to an apoptotic process coupled with a negligible cell proliferation. Cystathionine γ-lyase was diffusely up-regulated. Surviving aortic smooth muscle cells exhibited diverging phenotypes: in inner- and outer-media contractile cells prevailed, having higher contents of smooth-muscle-α-actin holoprotein (45-kDa) and of caspase-3-cleaved smooth-muscle-α-actin 25-kDa fragments; in mid-media, aortic smooth muscle cells exhibited a synthetic/secretor phenotype, down-regulating vimentin, but up-regulating glial fibrillary acidic protein, trans-Golgi network 46 protein, Jagged1 (172-kDa) holoprotein, and Jagged1's receptor Notch1. Extracellular soluble Jagged1 (42-kDa) fragments accumulated. Conclusions In SNSTAAs, there is a relentless aortic smooth muscle cells attrition caused by the up-regulated cystathionine γ-lyase. In mid-media, synthetic/secretor aortic smooth muscle cells intensify Jagged1/NOTCH1 signalling in the attempt to counterbalance the weakened aortic wall, due to aortic smooth muscle cells net loss and mechanical stress. Synthetic/secretor aortic smooth muscle cells are apoptosis-prone, and the accruing thrombin-cleaved Jagged1 fragments counteract the otherwise useful effects of Jagged1/NOTCH1 signalling, thus hampering tissue homeostasis/remodelling, and aortic smooth muscle cells adhesion, differentiation, and migration.

Preterm birth disrupts cerebellar development by affecting granule cell proliferation program and Bergmann glia.

Preterm birth is a leading cause of long-term motor and cognitive deficits. Clinical studies suggest that some of these deficits result from disruption of cerebellar development, but the mechanisms that mediate cerebellar abnormalities in preterm infants are largely unknown. Furthermore, it remains unclear whether preterm birth and precocious exposure to the ex-utero environment directly disrupt cerebellar development or indirectly by increasing the probability of cerebellar injury, including that resulting from clinical interventions and protocols associated with the care of preterm infants. In this study, we analyzed the cerebellum of preterm pigs delivered via c-section at 91% term and raised for 10 days, until term-equivalent age. The pigs did not receive any treatments known or suspected to affect cerebellar development and had no evidence of brain damage. Term pigs sacrificed at birth were used as controls. Immunohistochemical analysis revealed that preterm birth did not affect either size or numbers of Purkinje cells or molecular layer interneurons at term-equivalent age. The number of granule cell precursors and Bergmann glial fibers, however, were reduced in preterm pigs. Preterm pigs had reduced proliferation but not differentiation of granule cells. qRT-PCR analysis of laser capture microdissected external granule cell layer showed that preterm pigs had a reduced expression of Ccnd1 (Cyclin D1), Ccnb1 (Cyclin B1), granule cell master regulatory transcription factor Atoh1, and signaling molecule Jag1. In vitro rescue experiments identified Jag1 as a central granule cell gene affected by preterm birth. Thus, preterm birth and precocious exposure to the ex-utero environment disrupt cerebellum by modulating expression of key cerebellar developmental genes, predominantly affecting development of granule precursors and Bergmann glia.

JAG1 overexpression contributes to Notch1 signaling and the migration of HTLV-1-transformed ATL cells.

BACKGROUND: HTLV-1 is a retrovirus that infects over 20 million people worldwide and is responsible for the hematopoietic malignancy adult T cell leukemia (ATL). We previously demonstrated that Notch is constitutively activated in ATL cells. Activating genetic mutations were found in Notch; however, Notch signaling was also activated in the absence of genetic mutations suggesting the existence of other mechanisms. METHODS: We analyzed the expression of Notch receptor ligands in HTLV-I-transformed cells, ATL patient-derived cell lines, and fresh uncultured ATL samples by RT-PCR, FACS, and immunohistochemistry. We then investigated viral and cellular molecular mechanisms regulating expression of JAG1. Finally, using shRNA knock-down and neutralizing antibodies, we investigated the function of JAG1 in ATL cells. RESULTS: Here, we report the overexpression of the Notch ligand, JAG1, in freshly uncultured ATL patient samples compared to normal PBMCs. We found that in ATL cells, JAG1 overexpression relies upon the viral protein Tax and cellular miR-124a, STAT3, and NFATc1. Interestingly, our data show that blockade of JAG1 signaling dampens Notch1 downstream signaling and limits cell migration of transformed ATL cells. CONCLUSIONS: Our results suggest that targeting JAG1 can block Notch1 activation in HTLV-I-transformed cells and represents a new target for immunotherapy in ATL patients.

Long non-coding RNA MALAT1 interacts with miR-124 and modulates tongue cancer growth by targeting JAG1.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA (lncRNA), was the earliest discovered to be correlated with cancer and contributes to the initiation and development of several types of tumors. Dysregulation of MALAT1 expression is frequently observed in many types of cancer such as gastric cancer, esophageal squamous cell carcinoma and glioma. To date, the role of MALAT1 and the underlying mechanisms in tongue cancer development remain unclear. In the present study, we studied the influence of MALAT1 on tongue cancer cell lines and clinical tongue cancer samples so as to detect its function and the underlying mechanism. In the present study, lncRNA-MALAT1 was specifically upregulated in tongue cancer cell lines and overexpression promoted tongue cancer cell growth by targeting miR-124. Knockdown of MALAT1 suppressed the growth and invasion of human tongue cancer cells and inhibited metastasis in vitro and in vivo. In addition, miR-124-dependent jagged1 (JAG1) regulation was required for MALAT1-induced tongue cancer cell growth. Our data revealed that MALAT1 inhibited tongue cancer cell growth and metastasis through miR-124-dependent JAG1 regulation. In conclusion, we revealed that MALAT1 may play an oncogenic role by increasing proliferation and metastasis of tongue cancer and is a potential therapeutic target in human tongue cancer.

Jagged1 modulated tumor-associated macrophage differentiation predicts poor prognosis in patients with invasive micropapillary carcinoma of the breast.

OBJECTIVES: Invasive micropapillary carcinoma of the breast (IMPC) constitutes a unique and aggressive subtype of breast cancer. We aimed to evaluate the prognostic significance of the Jagged1 (a ligand of the Notch pathway) expression, and infiltration density of tumor-associated macrophages (TAMs) in patients with IMPC. METHODS: Jagged1 expression and CD163+, CD68+ macrophage infiltration were evaluated by immunohistochemistry in 222 tumor samples, and the clinical significance was analyzed. mRNA level of Jagged1 was analyzed by real time PCR in tumor tissues. RESULTS: The IMPC patients showed larger tumor size, more lymphatic invasion, higher expression levels of estrogen receptor (ER), increased Ki67 index, higher Jagged1 protein level, and denser infiltration of CD163+ macrophages compared to patients with invasive breast ductal carcinoma. In the IMPC cohort, positive Jagged1 expression was related to aggressive features including large tumor size, lymphatic invasion, and Ki67 overexpression. Statistical significance was found between CD163+ macrophage infiltration and Jagged1 expression levels. Cox regression analysis revealed that ER negativity, positive Jagged1 expression, and a high degree of CD163+ macrophage infiltration were independent prognostic factors for disease-free survival, and positive Jagged1 expression was an independent prognostic factor for overall survival. The level of Jagged1 mRNA was higher in tumor tissues of patients with IMPC. CONCLUSION: Jagged1, by modulating TAMs infiltration, is associated with a less favorable prognosis for patients with IMPC. Our results have important implications for therapies targeting Jagged1-Notch signaling and re-educating TAMs polarization for patients with IMPC.

Decreased TAp63 and ΔNp63 mRNA Levels in Most Human Pituitary Adenomas Are Correlated with Notch3/Jagged1 Relative Expression.

Despite recent advances in molecular genetics, the pituitary adenoma initiation, development, progress, and the molecular basis of their unique features are still poorly understood. In this sense, it is proposed that stem cell could be involved in pituitary adenoma tumorigenesis. It is suggested that TP63 has important functions in stem cells, and it may have interplay of TP63 and Notch and its ligand Jagged in this process. This study aimed to evaluate the distinct expression of TP63 isoforms (TAp63 and ΔNp63), as well as its correlation with Notch3 receptor and its ligand Jagged1 in human pituitary adenomas at the messenger RNA (mRNA) level. We included 77 pituitary adenoma tumor samples from patients who underwent surgical resection. The expression levels of TP63 isoforms (TAp63 and ΔNp63) and Notch3 and its ligand Jagged1 were evaluated by qRT-PCR using isoform-specific primers. We also evaluated proliferation index immunohistochemically using KI-67 antibody. The expression levels were associated with clinical outcomes, as age, gender, tumor size, and tumor subtype. In summary, we found that mRNA expression of both TP63 isoforms decreased in pituitary adenomas compared with normal pituitary control. On the other hand, there was an increase of relative Notch3 and Jagged1 mRNA expression in the majority of examined samples. The mRNA expression of three genes evaluated was correlated and statistically significantly. There was no significant association between gene expression and the analyzed clinical data. The current study has provided the first time evidence that Tap63 and ΔNp63 isoforms are underexpressed in most pituitary adenomas. These results are correlated with Notch3 and its ligand Jagged1 overexpression, corroborating previous studies pointing its antagonistic interactions.