Activation Of Notch Signaling Pathway Research Articles

Notch Signal Regulates Corneal Endothelial-to-Mesenchymal Transition

Endothelial-to-mesenchymal transition (EnMT) is a cell transformation process involved in both morphogenesis and pathogenesis. EnMT of corneal endothelial cells happens after endothelial injury and during ex vivo culture. Previous studies have shown that the transforming growth factor-β signaling pathway is involved in this transition. In this study, we found that rat corneal endothelial cells could spontaneously undergo EnMT during ex vivo culture. This change in rat corneal endothelial cells was associated with Notch signaling pathway activation after the first passage, which was blocked by the Notch inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). This inhibitor also prevented transforming growth factor β1-, β2-, and β3-induced EnMT and reversed transformed rat corneal endothelial cells to a normal phenotype. Furthermore, DAPT treatment blocked retrocorneal membrane formation in a rat corneal endothelium damage model. Our study indicates that the Notch signaling pathway is involved in the corneal EnMT process, which may be a novel therapeutic target for treating corneal endothelial fibrogenic disorders.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

AB0115 BFGF increases HES1 and HEY1 expressions in murine chondrocytes in vitro

BackgroundDuring osteoarthritis (OA), articular chondrocytes undergo cellular changes, such as proliferation and dedifferenciation, reminiscent of growth plate (GP) terminal différentiation. Among the factors and the signaling molecules which control this...

Abstract 4104: Netrin-1 promotes the invasiveness and survival of human glioblastoma cells.

Abstract Glioblastoma multiforme (GBM) is the most severe human brain cancer, which has no efficient treatment. The main reason for the lethality of GBM is its resistance to all current therapies and highly invasive. According to microarray data on Oncomine database netrin-1 is upregulated in glioblastoma tumors. Netrin-1 is a secreted laminin related extracellular matrix protein. Its main function is to regulate the axon guidance during embryogenesis. Recently it has been observed to act as a survival factor for different forms of cancer such as aggressive neuroblastoma, metastatic breast cancer and non small cell lung cancer. It also promotes tumorigenesis of colorectal cancer. Based on these observations we explored the possibility whether netrin-1 plays a role in human GBM. We analyzed the effects of netrin-1 expression on human GBM cells. According to Matrigel invasion assays overexpression of netrin-1 increased the invasiveness whereas partial knock-down of netrin-1 by shRNAs reduced the invasiveness of human GBM cells. On the other hand, complete knock-down of netrin-1 led to apoptotic death of those cells. To further analyze netrin-1 signaling in human GBM we performed a mass spectrometry screen to identify binding partners of netrin-1. We identified Notch as a novel receptor for netrin-1. In addition, we observed that netrin-1 overexpression increased and knockdown decreased Notch activation. The role of Notch signaling regarding the aggressiveness of various cancers has been controversial. Our results confirm that increased Notch signaling increases glioblastoma cell invasiveness, and that netrin-1 increases Notch signaling. Further, we identified here a recombinant netrin-1 fragment that inhibits Notch signaling by blocking the Notch signaling complex to the cell surface. Current studies provide new mechanistic information on the tumorigenesis of GBM. Activation of Notch signaling pathway has been linked to the initiation of EMT which is essential for cancer cell invasion. Our results suggest that netrin-1 regulates GBM invasion by activating Notch signaling. The inhibition of netrin-1 signaling may prove out to be a treatment option for human glioblastoma. Citation Format: Jorma K. Keski-Oja, Irene Ylivinkka, Yizhou Hu, Tuula Nyman, Marko Hyytiainen. Netrin-1 promotes the invasiveness and survival of human glioblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4104. doi:10.1158/1538-7445.AM2013-4104

Notch pathway activation targets AML-initiating cell homeostasis and differentiation

Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia.

Protective Effect of Catalpol on Myocardium in Rats with Isoprenaline-Induced Myocardial Infarcts via Angiogenesis through Endothelial Progenitor Cells and Notch1 Signaling Pathway

Protective effect of catalpol on myocardium was studied in relation to endothelial progenitor cells, Notch1 signaling pathway and angiogenesis in rats with isoprenaline (INN)-induced acute myocardial infarcts. To analyze the pathological status and impact of catalpol on the rats, 3 weeks after intragastric gavage, the animals were verified for myocardial infarcts with electrocardiogram and measured for enzyme activity of lactate dehydrogenase (LDH), malondialdehyde (MDA), creatine kinase (CK) and superoxide dismutase (SOD) in myocardium, and further analyzed using HE and TTC staining, as well as visual examination of infarct area. Flow cytometry study of endothelial progenitor cells (EPCs) indicated that the EPCs were mobilized during infarction. The roles of Notch1 signaling pathway in angiogenesis of the infracted animals were studied using immunohistochemistry analysis of RBPjκ and Western blot analysis of Notch1 and Jagged1. Our results obtained from the rats treated with catalpol, positive drug and control showed that catalpol could protect rats from infarction probably by mobilization of EPCs and activation of Notch1 signaling pathway.

The periodontal pathogen Porphyromonas gingivalis changes the gene expression in vascular smooth muscle cells involving the TGFbeta/Notch signalling pathway and increased cell proliferation

BackgroundPorphyromonas gingivalis is a gram-negative bacterium that causes destructive chronic periodontitis. In addition, this bacterium is also involved in the development of cardiovascular disease. The aim of this study was to investigate the effects of P. gingivalis infection on gene and protein expression in human aortic smooth muscle cells (AoSMCs) and its relation to cellular function.ResultsAoSMCs were exposed to viable P. gingivalis for 24 h, whereafter confocal fluorescence microscopy was used to study P. gingivalis invasion of AoSMCs. AoSMCs proliferation was evaluated by neutral red assay. Human genome microarray, western blot and ELISA were used to investigate how P. gingivalis changes the gene and protein expression of AoSMCs. We found that viable P. gingivalis invades AoSMCs, disrupts stress fiber structures and significantly increases cell proliferation. Microarray results showed that, a total of 982 genes were identified as differentially expressed with the threshold log2 fold change > |1| (adjust p-value <0.05). Using bioinformatic data mining, we demonstrated that up-regulated genes are enriched in gene ontology function of positive control of cell proliferation and down-regulated genes are enriched in the function of negative control of cell proliferation. The results from pathway analysis revealed that all the genes belonging to these two categories induced by P. gingivalis were enriched in 25 pathways, including genes of Notch and TGF-beta pathways.ConclusionsThis study demonstrates that P. gingivalis is able to invade AoSMCs and stimulate their proliferation. The activation of TGF-beta and Notch signaling pathways may be involved in the bacteria-mediated proliferation of AoSMCs. These findings further support the association between periodontitis and cardiovascular diseases.

Progress in understanding the relationship between Notch signaling pathway and hepatic stellate cell activation

Progress in understanding the relationship between Notch signaling pathway and hepatic stellate cell activation

A Clinical Study of Bone Marrow Stromal Cell Therapy in Patients with Refractory Cgvhd by Down-Regulating the Jagged2 Gene

AbstractAbstract 4673Refractory chronic GVHD (cGVHD) is an important complication after allogeneic hematopoietic SCT and is prognostic of poor outcome. Bone Marrow Stromal Cells (MSCs) are involved in tissue repair and modulating immune responses in vitro and in vivo. MSCs as salvage treatment for refractory cGVHD have been reported in our previous study, however, the possible mechanism have yet not to be determined.Between November 2006 and November 2010, 18 patients were diagnosed with refractory cGVHD, 8 patients were treated with in vitro expanded BM-derived MSCs as a compassionate treatment for refractory cGVHD, 10 patients that did not receive BMSCs treatment were control group. The median MSC dose given was 0.6×106/kg body weight. MSCs were harvested fresh from culture and administered to the patients by intravenous infusions over 30 minutes. The median time of MSC administrations was 3 (range, 2–6). The response was assessed monthly after BMSCs treatment, and the total follow-up period was 6 months. The organ response and the overall response were used to determine the therapeutic efficacies of MSC for refractory cGVHD. The expression of the Jagged2 gene of peripheral blood mononuclear cells in patients at the assessment points were analyzed using the TaqMan real-time polymerase chain reaction, with ABL mRNA expression levels as an internal reference.After BMSCs treatment, a total of 6 patients (75%) had an overall response (PR n=6), and 2 patients had a minor partial response (mPR n=2). The expression levels of Jagged2 mRNA in these cases at the diagnosis of refractory cGVHD were significantly increased, compared with none cGVHD patients (23.94%±18.68% vs 3.76%±1.50%, P < 0.05), and the copies of Jagged2 mRNA in BMSC treatment responsed patients’ peripheral blood were significantly reduced (5.15%±3.25%, P <0.05), while Jagged2 mRNA expression levels of the control group were no significant difference (P> 0.05).Our pilot study showed that Jagged2 gene reproduction upregulated when the cGVHD is active, so, dynamic monitoring of Jagged2 mRNA expression may have the potential effect on predicting the activity of chronic graft-versus-host disease. Mechanism of Bone marrow stromal cells to treat refractory cGVHD may be related to down-regulation of donor T cells Notch ligand Jagged2 gene expression, which suppression of T cell Notch signaling pathway activation, thus inducing immune tolerance. Disclosures:No relevant conflicts of interest to declare.

A Novel Mechanism for Sevoflurane Preconditioning-induced Neuroprotection

A Novel Mechanism for Sevoflurane Preconditioning-induced Neuroprotection

Activation of Canonical Notch Signaling Pathway Is Involved in the Ischemic Tolerance Induced by Sevoflurane Preconditioning in Mice

A wealth of evidence has demonstrated that sevoflurane preconditioning induces brain ischemic tolerance, but the mechanism remains poorly understood. This study was designed to investigate the role of canonical Notch signaling in the neuroprotection induced by sevoflurane preconditioning in a mouse model. C57BL/6 mice were pretreated with 1-h sevoflurane exposure at a dose of 2.5% for 5 consecutive days. Twenty-four hours after the last exposure, all mice were subjected to focal cerebral ischemia by right middle cerebral artery occlusion for 60 min. Neurobehavioral scores, brain infarct volumes, and cellular apoptosis were determined at 72 h after reperfusion (n = 10 per group). The activation of Notch signaling was evaluated (n = 5 per group), and its role in ischemic tolerance was assessed by intraperitoneal administration of γ-secretase inhibitor DAPT (100 mg/kg, n = 10 per group) and conditional Notch-RBP-J knockout technique (n = 8 per group). Sevoflurane preconditioning reduced brain infarct volumes (42.5%), improved neurologic outcomes (P < 0.01 vs. control), and attenuated neuronal cell apoptosis (cells positive for terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling reduced to 21.2%). The expression of Notch1 intracellular domain (1.35 folds) and the transcriptions of Hes1 (1.95 times) and Hes5 (1.48 times) were up-regulated. DAPT augmented the brain infarcts (1.64-fold) and decreased neurologic scores (P = 0.43 vs. sevoflurane) in sevoflurane-preconditioned mice. Brain infarct volumes, neurobehavioral scores, and apoptotic cell numbers showed no significance between Notch knockout mice with sevoflurane preconditioning and wild-type mice without preconditioning. Sevoflurane preconditioning-induced protective effects against transient cerebral ischemic injuries are mediated by the activation of canonical Notch signaling pathway in mice.

Involvement of Notch signaling pathway in regulating IL-12 expression via c-Rel in activated macrophages

Macrophages play an important role both in innate and adaptive immune responses. Treatment with interferon (IFN) γ together with lipopolysaccharide (LPS) activates pro-inflammatory macrophages which secrete various pro-inflammatory cytokines including IL-12. IL-12 promotes a Th1 type immune response by directly controlling the differentiation of CD4+ T helper 1 cells. Activation of Notch signaling pathway was reported in activated macrophages but the involvement of this signaling pathway in IL-12 expression has not been documented. In this study, we investigated the role of Notch signaling in regulating expression of the IL-12/IL-23 subunit, IL-12p40. Using a gamma-secretase inhibitor (GSI) to inhibit Notch signaling, we observed a profound decrease in il12p40 mRNA levels and IL-12p70 secretion upon IFNγ/LPS stimulation. On the other hand, overexpression of activated form of Notch1 in activated RAW264.7 macrophage-like cell lines significantly increased the level of il12p40 mRNA. GSI treatment did not affect the expression of irf5, a master regulator of il12p40 transcription in macrophages. Detailed analysis of the signaling cascades that were affected by this inhibition showed that c-Rel nuclear translocation was inhibited and Erk1/2 activation was compromised by GSI treatment. Addition of exogenous tumor necrosis factor (TNF) α only partially rescued the expression of il12p40 in the presence of GSI. Unexpectedly, inhibition of Notch signaling using a dominant negative (DN) Mastermind-like (MAML) transcription co-activator, did not affect c-Rel nuclear localization upon activation or il12p40 mRNA levels, suggesting that the transcriptional activity of Notch signaling is dispensable for the activation of c-Rel. These results strongly suggest that Notch signaling in activated macrophages is involved in regulating the expression of il12p40 directly via c-Rel and indirectly via TNFα production.

Notch Signaling Pathway and Human Placenta

Notch signaling was evolutionarily conserved and critical for cell-fate determination, differentiation and many other biological processes. Growing evidences suggested that Notch signaling pathway played an important role in the mammalian placental development. All of the mammalian Notch family proteins had been identified in human placenta except Delta-like 3, which appeared to affect the axial skeletal system. However the molecular mechanisms that regulated the Notch signaling pathway remained largely unknown in human placenta. Therefore, additional research was needed to investigate expression pattern of Notch family members and the mechanisms for activation of Notch signaling pathway in human placenta, which might help elucidate the roles of Notch signaling pathway in human placentation. This review would focus on the roles of Notch receptors and ligands in the human placental trophoblasts function and placental angiogenesis. It might hopefully provide perspectives for future research about human placentation of pregnancy complicated by preeclampsia and other placenta associated diseases.

Fate Restriction and Multipotency in Retinal Stem Cells

Stem cells have the capacity to both self-renew and generate postmitotic cells. Long-term tracking of individual clones in their natural environment constitutes the ultimate way to validate postembryonic stem cells. We identify retinal stem cells (RSCs) using the spatiotemporal organization of the fish retina and follow the complete offspring of a single cell during the postnatal life. RSCs generate two tissues of the adult fish retina, the neural retina (NR) and the retinal-pigmented epithelium (RPE). Despite their common embryonic origin and tight coordination during continuous organ growth, we prove that NR and RPE are maintained by dedicated RSCs that contribute in a fate-restricted manner to either one or the other tissue. We show that in the NR, RSCs are multipotent and generate all neuron types and glia. The clonal origin of these different cell types from a multipotent NSC has far-reaching implications for cell type and tissue homeostasis.

Activation of the Canonical Notch Signaling Pathway in the Hematopoietic Microenvironment Is Required to Maintain Normal Hematopoiesis

Abstract 723Recent studies demonstrated that the causes of Myeloproliferative syndromes (MPS) is not limited to factors intrinsic to hematopoietic cells, but include also non-cell autonomous mechanisms. Here, we demonstrate that loss of CSL-dependent Notch signaling in the hematopoietic microenvironment leads to a lethal myeloproliferative disorder. Following pIpC induction, Mx1-Cre+/RBP-Jlox/lox mice showed a significantly increase in granulocyte/macrophage progenitors and granulocytes in bone marrow (BM), peripheral blood, spleen and liver and died by week 16–20. Survival at 5 months was 12% for the RBPJ KO mice compared to 100% for the control mice. Transplant studies revealed that the myeloproliferative disorder occurred rapidly when WT-hematopoietic cells were transplanted into MX1-Cre+/RBP-Jlox/lox-recipients, whereas WT-recipients of MX1-Cre+/RBP-Jlox/lox hematopoietic cells did not show onset of diseases at 6 months of follow-up. Furthermore, the disease was not transplantable in WT-recipients, indicating that abnormal Notch signaling in the microenvironment is critical for the development and sustainment of MPS. Transplants conducted with Lys/GFP HSC as WT donor cells, showed that transplant of myeloid progenitors (LK) are sufficient for the development of disease, as documented by rapid expansion of LK-Lys/GFP cells into MX1-Cre+/RBP-Jlox/lox recipients by intravital microscopy of the BM niche. Levels of hematopoietic cytokines were not significantly different in MX1-Cre+/RBP-Jlox/lox recipients prior to transplantation. Histological analysis of BM and spleen showed increased microvascular density and increased CD31+ EC by IHC. To dissect the mechanisms, we analyzed different cellular components of the microenvironment. Mx1Cre driven deletion of RBP-J occurred in osteoblasts, endothelial progenitors and mesenchymal stem cells. Co-culture experiments of HSC with these different cell types suggested cooperative effects in altering proliferation and differentiation of myeloid cells. Crosses of RBP-Jlox/lox mice with mice bearing cell type promoter specific Cre are ongoing to determine the cellular type(s) critical for the myeloid expansion and to identify downstream molecular mediators. These studies will provide further insights on the role of the microenvironment in contributing to myeloid proliferative diseases and leukemia. Disclosures:No relevant conflicts of interest to declare.

HIF-1α is critical for hypoxia-mediated maintenance of glioblastoma stem cells by activating Notch signaling pathway

Hypoxia induces the expansion of glioblastoma stem cells (GSCs), but the mechanism underlying it is still unclear. Here, we supply evidence that hypoxia-inducible factor-1α (HIF-1α) induced activation of Notch pathway is essential for hypoxia-mediated maintenance of GSC. Either depletion of HIF-1α or inactivation of Notch signaling partly inhibits the hypoxia-mediated maintenance of GSC. Further data suggest a role for HIF-1α in the interaction and stabilization of intracellular domain of Notch (NICD), and activation of Notch signaling. The mRNA level of HIF-1α and Notch target gene FABP7 was elevated in GSC. And the STAT3 pathway responsible for the HIF-1α gene transcription, the phosphatidylinositol 3-kinase-Akt and ERK1/2, both of which are contributed to HIF-1α protein translation, are also preferentially activated in GSC. Inhibition of these pathways partly reduces the hypoxia-induced activation of the Notch pathway and subsequent GSC maintenance. Taken together, our findings suggest that HIF-1α requires Notch pathway to drive the maintenance of GSC. The activated regulation of HIF-1α makes GSC more sensitive to hypoxia-mediated maintenance. These findings enhance our understanding of mechanism of hypoxia-mediated GSC expansion and provide HIF-1α as an attractive target for glioblastoma therapy.

Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

High levels of pro-angiogenic factors, leptin, IL-1, Notch and VEGF (ligands and receptors), are found in breast cancer, which is commonly correlated with metastasis and lower survival of patients. We have previously reported that leptin induces the growth of breast cancer and the expression of VEGF/VEGFR-2 and IL-1 system. We hypothesized that Notch, IL-1 and leptin crosstalk outcome (NILCO) plays an essential role in the regulation of leptin-mediated induction of proliferation/migration and expression of pro-angiogenic molecules in breast cancer. To test this hypothesis, leptin's effects on the expression and activation of Notch signaling pathway and VEGF/VEGFR-2/IL-1 were determined in mouse (4T1, EMT6 and MMT) breast cancer cells. Remarkably, leptin up-regulated Notch1-4/JAG1/Dll-4, Notch target genes: Hey2 and survivin, together with IL-1 and VEGF/VEGFR-2. RNA knockdown and pharmacological inhibitors of leptin signaling significantly abrogated activity of reporter gene-luciferase CSL (RBP-Jk) promoter, showing that it was linked to leptin-activated JAK2/STAT3, MAPK, PI-3K/mTOR, p38 and JNK signaling pathways. Interestingly, leptin upregulatory effects on cell proliferation/migration and pro-angiogenic factors Notch, IL-1 and VEGF/VEGFR-2 were abrogated by a γ-secretase inhibitor, DAPT, as well as siRNA against CSL. In addition, blockade of IL-1R tI inhibited leptin-induced Notch, Hey2 and survivin as well as VEGF/VEGFR-2 expression. These data suggest leptin is an inducer of Notch (expression/activation) and IL-1 signaling modulates leptin effects on Notch and VEGF/VEGFR-2. We show for the first time that a novel unveiled crosstalk between Notch, IL-1 and leptin (NILCO) occurs in breast cancer. Leptin induction of proliferation/migration and upregulation of VEGF/VEGFR-2 in breast cancer cells were related to an intact Notch signaling axis. NILCO could represent the integration of developmental, pro-inflammatory and pro-angiogenic signals critical for leptin-induced cell proliferation/migration and regulation of VEGF/VEGFR-2 in breast cancer. Targeting NILCO might help to design new pharmacological strategies aimed at controlling breast cancer growth and angiogenesis.

Activated K-ras and INK4a/Arf Deficiency Cooperate During the Development of Pancreatic Cancer by Activation of Notch and NF-κB Signaling Pathways

BackgroundPancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, suggesting that novel strategies for the prevention and treatment of PDAC are urgently needed. K-ras mutations are observed in >90% of pancreatic cancer, suggesting its role in the initiation and early developmental stages of PDAC. In order to gain mechanistic insight as to the role of mutated K-ras, several mouse models have been developed by targeting a conditionally mutated K-rasG12D for recapitulating PDAC. A significant co-operativity has been shown in tumor development and metastasis in a compound mouse model with activated K-ras and Ink4a/Arf deficiency. However, the molecular mechanism(s) by which K-ras and Ink4a/Arf deficiency contribute to PDAC has not been fully elucidated.Methodology/Principal FindingsTo assess the molecular mechanism(s) that are involved in the development of PDAC in the compound transgenic mice with activated K-ras and Ink4a/Arf deficiency, we used multiple methods, such as Real-time RT-PCR, western blotting assay, immunohistochemistry, MTT assay, invasion, EMSA and ELISA. We found that the deletion of Ink4a/Arf in K-rasG12D expressing mice leads to PDAC, which is in part mediated through the activation of Notch and NF-κB signaling pathways. Moreover, we found down-regulation of miR-200 family, which could also play important roles in tumor development and progression of PDAC in the compound transgenic mice.Conclusions/SignificanceOur results suggest that the activation of Notch and NF-κB together with the loss of miR-200 family is mechanistically linked with the development and progression of PDAC in the compound K-rasG12D and Ink4a/Arf deficient transgenic mice.

Abstract 4086: Notch, IL-1 and leptin crosstalk outcome (NILCO) is critical for leptin regulation of VEGF/VEGFR-2 in breast cancer

Abstract Elevated levels of leptin and its deregulated signals have been strongly associated to obesity-related cancers, including breast cancer. Higher levels of leptin and leptin receptor, OB-R, are expressed by breast cancer cells, which commonly correlated with metastasis and lower survival of breast cancer patients. We have previously reported that leptin signaling induced the growth of breast cancer mainly through the up-regulation of pro-angiogenic molecules, i.e., VEGF/VEGFR-2, IL-1. Disruption of leptin signaling markedly reduced the growth of tumors and the expression of VEGF/VEGFR-2 in syngeneic and human breast cancer xenograft mouse models (ER+ and ER-). We speculate leptin interacts with Notch (essential for tumor progression and angiogenesis) in breast cancer cells through complex molecular mechanisms. We hypothesized that a crosstalk between leptin and Notch signaling could impact the expression of pro-angiogenic molecules in breast cancer. Methods: To test this hypothesis leptin-mediated activation of Notch signaling pathway (Notch1,3,4 NICD and CSL) and VEGF/VEGFR2 expression were determined in mouse (4T1, EMT6 and MMT) and human breast cancer cell lines (MCF-7 and MDA-MB-231) under normoxic conditions. Results: Leptin upregulates Notch receptors/ligands and also Notch-target genes, Hey2 and survivin in breast cancer cells. Notably, these leptin effects were more evident in triple negative breast cancer cells (MDA-MB231). Leptin-induction of CSL promoter activity was linked to multiple signaling pathways (MAPK, PI-3K/mTOR, PKC, p38 and JNK). Interestingly, leptin upregulatory effects on pro-angiogenic factors IL-1, VEGF/VEGFR-2 and Notch were significantly abrogated by a γ-secretase inhibitor, DAPT, as well as siRNA against CSL. In addition, IL-1 tI antibody inhibits leptin induced Notch, Notch target genes, Hey2 and survivin as well as VEGF/VEGFR-2 expression. Conclusions: These data suggest leptin-induced IL-1 signaling is related to leptin effects on Notch signaling. Our results further indicate that a novel unveiled crosstalk between Notch, IL-1 and leptin occurs in breast cancer. Indeed, leptin up-regulation of pro-angiogenic molecules: Notch, VEGF/VEGFR-2, IL-1 requires the activation of Notch signaling in breast cancer cells. Therefore, the integration of developmental, pro-inflammatory and pro-angiogenic signals (Notch, IL-1 and leptin crosstalk outcome (NILCO)] seems to be critical for leptin regulation of VEGF/VEGFR-2 in breast cancer. This novel finding might help to design new pharmacological strategies aimed at controlling breast cancer growth and angiogenesis, especially in triple negative breast cancer patients. [This work was supported in part by NIH/NCI1SC1CA138658-02; NIH/ARRA/3SC1CA138658-02S1and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP)]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4086. doi:10.1158/1538-7445.AM2011-4086

Expression of the Active Notch1 Decreases MTC Tumor Growth In Vivo

Human medullary thyroid cancer (MTC) is a neuroendocrine (NE) tumor, derived from thyroid C-cells. Besides surgery, there are no curative therapies for MTC. This emphasizes the need for the development of new therapies. In MTC, Notch1 signaling pathway is absent and Notch1 activation in MTC-TT cells has been shown to reduce growth and NE markers in vitro. While the in vitro studies will provide insight into the potential mechanisms by which Notch inhibits growth, only by in vivo model one can recreate the conditions found in patients with MTC and assess effects on metastatic potential and microscopic disease. Doxycycline inducible TT-NOTCH1 cells were utilized in a murine subcutaneous xenograft model to study tumor development and growth. Doxycycline was used to induce the expression of Notch1 in these tumors. Measurements of tumor volume showed that doxycycline treated mice had slower tumor growth than control mice. Western blot analysis of tumor lysates demonstrated activation of Notch1 protein only in doxycycline treated mice suggesting that active Notch1 slowed tumor growth. Furthermore, this activation led to a significant reduction in the levels of achaete-scute complex-like1 and chromogranin A important NE markers. Based on these data, activation of Notch signaling pathway could be a therapeutic strategy to treat patients with MTC.