Notch Target Genes Hey1 Research Articles

Doxorubicin Inhibits Proliferation of Osteosarcoma Cells Through Upregulation of the Notch Signaling Pathway.

Doxorubicin plays a major role in the treatment of osteosarcoma disorders. The Notch signaling pathway exerts various biological functions, including cell proliferation, differentiation, and apoptosis. In the present study, we investigated the effects of different doses of doxorubicin on proliferation and apoptosis of osteosarcoma cells with or without Notch signaling. Results found that cellular viability was downregulated while caspase 3 activity and expression were promoted in osteosarcoma cells following treatment with various doses of doxorubicin for 24, 48, and 72 h, and the effects showed a dose- and time-dependent manner. Furthermore, it was found that various doses of doxorubicin activated the Notch signaling pathway, shown by the elevated expression of Notch target genes NOTCH1, HEY1, HES1, AND HES5. It was further proved that, after small interfering RNA (siRNA)-mediated knockdown of Notch, the effects of doxorubicin on the viability and apoptosis of osteosarcoma cells were significantly reduced. It was indicated that doxorubicin treatment reduced the proliferation and promoted the apoptosis of osteosarcoma cells, and this effect was mediated by the Notch signaling pathway.

Abstract 4240: TORC inhibition enriches for a cancer stem cell-like population with FGFR-dependent Notch1 activation

Abstract Background. Cancer stem cells (CSCs) are associated with metastatic recurrences and poor prognosis in triple negative breast cancer (TNBC) patients. Genomic and proteomic data suggest that >30% of TNBC harbor PI3K/mTOR pathway aberrations. Furthermore, PI3K/mTOR inhibitors have shown antitumor activity in preclinical TNBC models. However, resistance to PI3K/mTOR inhibitors in breast and ovarian cancer has been recently shown. We hypothesized that resistance to TORC inhibition in TNBC is driven by a CSC-like population and targeting this population can improve antitumor action of TORC inhibitors. Methods and Results. Treatment of TNBC cells, SUM159 (PIK3CA-H1047L), BT549 (PTEN-del), and MDA468 (PTEN-del), with the PI3K/mTOR inhibitor BEZ235 or the TORC1/2 inhibitor MLN128 reduced growth. However, cells that survived BEZ235 and MLN128 treatment displayed CSC properties as assessed by FACS analysis for CSC markers and mammosphere formation. Using a Stem Cell-specific PCR Array and qRT-PCR, Notch1, Jagged1 and FGF1 mRNA levels were elevated in BEZ235 and MLN128-treated TNBC cells. Transient and long-term treatment with BEZ235 and MLN128 also increased expression of the active Notch intracellular domain (NICD), Notch target genes Hes1, Hey1 and c-Myc, and NICD-luciferase reporter activity. Nanostring analysis of post-chemotherapy primary breast cancers displayed increased expression of CSC markers (ALDH1A1 and CD44), Notch1, and Jagged1. Cells surviving the combination of the chemotherapeutic paclitaxel with BEZ235 or MLN128 displayed increased NICD and CSC-like properties. Similar changes were observed in OVCAR8 ovarian cancer cells treated with MLN128 ± cisplatin. Notch1 siRNA and the γ-secretase inhibitor (GSI) DAPT abrogated BEZ235- and MLN128-mediated enrichment of CSC markers and mammosphere formation. Treatment of SUM159 and MDA468 xenografts established in nude mice with MLN128 and DAPT did not significantly decrease tumor growth compared to MLN128 alone. However, MLN128 and DAPT-treated tumors displayed decreased tumorigenicity compared to MLN128-treated tumors as assessed by an in vivo limiting dilution assay. Furthermore, TNBC cells stably expressing mTOR shRNA exhibited increased NICD levels, CSC markers and tumorigenicity in vivo which was reduced with DAPT. With the increased FGF1 levels observed with BEZ235 and MLN128 treatment, we examined the effect of FRS2 siRNA and the FGFR1 kinase inhibitor lucitanib. Both approaches resulted in a reduction of the CSCs and NICD expression in MLN128-treated cells. Conclusions. These data suggest that treatment of TNBC harboring PI3K pathway aberrations with TORC1/2 inhibitors results in decreased tumor growth but may spare a drug-resistant CSC population with FGFR-Notch signaling. Thus, combination of Notch and TORC1/2 inhibitors in addition to chemotherapy may effectively decrease primary tumor growth and prevent recurrences in TNBC patients. Citation Format: Neil E. Bhola, Valerie M. Jansen, Carlos L. Arteaga. TORC inhibition enriches for a cancer stem cell-like population with FGFR-dependent Notch1 activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4240. doi:10.1158/1538-7445.AM2015-4240

Qingyihuaji Formula Inhibits Pancreatic Cancer and Prolongs Survival by Downregulating Hes-1 and Hey-1.

The dire prognosis of pancreatic cancer has not markedly improved during past decades. The present study was carried out to explore the effect of Qingyihuaji formula (QYHJ) on inhibiting pancreatic cancer and prolonging survival in related Notch signaling pathway. Proliferation of pancreatic cancer cells (SW1990 and PANC-1) was detected by MTT assay at 24, 48, and 72 h with exposure to various concentrations (0.08–50 mg/mL) of QYHJ water extract. Pancreatic tumor models of nude mice were divided into three groups randomly (control, QYHJ, and gemcitabine). mRNA and protein expression of Notch target genes (Hes-1, Hey-1, Hey-2, and Hey-L) in dissected tumor tissue were detected. Results showed that proliferation of SW1990 cells and PANC-1 cells was inhibited by QYHJ water extract in a dose-dependent and time-dependent manner. QYHJ effectively inhibited tumor growth and prolonged survival time in nude mice. Expression of both Hes-1 and Hey-1 was decreased significantly in QYHJ group, suggesting that Hes-1 and Hey-1 in Notch signaling pathway might be potential targets for QYHJ treatment. This research could help explain the clinical effectiveness of QYHJ and may provide advanced pancreatic cancer patients with a new therapeutic option.

Hes1, an important gene for activation of hepatic stellate cells, is regulated by Notch1 and TGF-β/BMP signaling.

To determine the role of Notch1 and Hes1 in regulating the activation of hepatic stellate cells (HSCs) and whether Hes1 is regulated by transforming growth factor (TGF)/bone morphogenetic protein (BMP) signaling. Immunofluorescence staining was used to detect the expression of desmin, glial fibrillary acidic protein and the myofibroblastic marker α-smooth muscle actin (α-SMA) after freshly isolated, normal rat HSCs had been activated in culture for different numbers of days (0, 1, 3, 7 and 10 d). The expression of α-SMA, collagen1α2 (COL1α2), Notch receptors (Notch1-4), and the Notch target genes Hes1 and Hey1 were analyzed by reverse transcriptase-polymerase chain reaction. Luciferase reporter assays and Western blot were used to study the regulation of α-SMA, COL1α1, COL1α2 and Hes1 by NICD1, Hes1, CA-ALK3, and CA-ALK5 in HSC-T6 cells. Moreover, the effects of inhibiting Hes1 function in HSC-T6 cells using a Hes1 decoy were also investigated. The expression of Notch1 and Hes1 mRNAs was significantly down-regulated during the culture of freshly isolated HSCs. In HSC-T6 cells, Notch1 inhibited the promoter activities of α-SMA, COL1α1 and COL1α2. On the other hand, Hes1 enhanced the promoter activities of α-SMA and COL1α2, and this effect could be blocked by inhibiting Hes1 function with a Hes1 decoy. Furthermore, co-transfection of pcDNA3-CA-ALK3 (BMP signaling activin receptor-like kinase 3) and pcDNA3.1-NICD1 further increased the expression of Hes1 compared with transfection of either vector alone in HSC-T6 cells, while pcDNA3-CA-ALK5 (TGF-β signaling activin receptor-like kinase 5) reduced the effect of NICD1 on Hes1 expression. Selective interruption of Hes1 or maintenance of Hes1 at a reasonable level decreases the promoter activities of α-SMA and COL1α2, and these conditions may provide an anti-fibrotic strategy against hepatic fibrosis.

The Notch Target Genes Hey1 and Hes7 Transcriptionally Suppress Gli1 Expression and Hedgehog Signaling in Hodgkin-Reed/Sternberg Cells of Classical Hodgkin Lymphoma: A Novel Mechanism of Drug Resistance

Tumor cell proliferation and survival of Hodgkin/Reed-Sternberg (HRS) cells are triggered through Jagged1 ligand-induced Notch1 signaling via homotypic and heterotypic cell-cell interactions in classical Hodgkin lymphoma. The developmental pathway Notch partly mediates its effects in HRS cells by stimulation of alternative NF-kB signaling. We further demonstrated that high-level expression of the essential Notch coactivator Mastermind-like 2 and downregulation of the Notch inhibitor Deltex1 contribute to aberrant activation of Notch signaling in HRS cells. Our data suggested that targeting the Notch pathway is a rational treatment strategy in classical Hodgkin lymphoma. In this study we analyzed Notch inhibition by use of the gamma secretase inhibitor GSI XII in a Hodgkin lymphoma xenotransplantation model. To this end the HRS cell line L540cy (1 x 107 cells/per mouse) was transplanted into NOD/SCID mice. After tumor growth (0.3 cm³ mean tumor volume) mice were treated daily with increasing doses of GSI XII (5-10 mg/kg). Surprisingly, L540cy cells were completely drug-resistant in vivo in contrast to high GSI XII sensitivity in vitro. To dissect potential mechanisms of drug resistance we performed human StellARrayTM quantitative polymerase chain reaction (qPCR) arrays to analyze Notch target genes in GSI XII-treated compared to untreated L540cy cells. Interestingly, inhibition of Notch activity resulted in strong mRNA upregulation of the transcription factor glioma-associated oncogene 1 (Gli1), a final effector of the developmental signaling pathway Hedgehog (HH). Chromatin immunoprecipitation (ChIP) further revealed that both negative regulatory Notch target proteins Hey1 and Hes7 directly bind three different N-boxes present in the GLI1 first intron to suppress GLI1 mRNA expression in untreated L540cy cells. In general, the HH pathway is activated through ligand binding of secreted Sonic Hedgehog (SHH). As a result Gli transcription factors translocate to the nucleus and induce target gene expression such as GLI1 or CCND1. Despite high secretion of SHH by HRS cells after two days in culture (conditioned medium), HH signaling was inactive in untreated L540cy cells. Only after release of the negative regulatory Notch targets of the hairy and enhancer of split (HES) family through Notch inhibition and concomitant increase of Gli1 expression, HH signaling was activated by SHH. HH signaling mediated drug resistance of L540cy cells in conditioned medium compared to fresh medium (SHH negative) and thereby compensated for reduced Notch activity in vitro. We hypothesized that this mechanism might contribute to GSI XII drug resistance in vivo. To proof our hypothesis we coinhibited the Notch and HH pathways in L540cy cells. As expected inhibition of the HH pathway alone by use of cyclopamine did not significantly reduce growth of L540cy cells. However, simultaneous targeting of L540cy tumors through GSI XII and cyclopamine efficiently controlled tumor cell growth. Our data indicate a first molecular link between Notch and HH in HRS cells mediating drug resistance. We suggest inhibition of both developmental pathways for effective HRS tumor growth control. DisclosuresNo relevant conflicts of interest to declare.

Notch- and TNFα-Activated Signaling Pathways Mediate Osteocyte Apoptosis Triggered By Multiple Myeloma Cells

Osteocytes comprise 95% of all bone cells and are central regulators of bone homeostasis and skeletal integrity. However the role of osteocytes in MM bone disease is unknown. We have previously shown that interactions with multiple myeloma cells have a profound effect on osteocytic gene expression, increasing Sost and RANKL transcripts and decreasing OPG. More recently, we and others have shown that myeloma cells increase the prevalence of osteocyte apoptosis, which might contribute to MM induced bone disease by increasing resorption in specific areas of bone. However, the mechanisms responsible for and the potential for targeting osteocyte apoptosis in myeloma are unknown. Osteocytes extensively communicate with each other and with cells on the bone surface and in the marrow, through cytoplasmic connections that run within canaliculi, which allows direct cell-to-cell contact and the distribution of secreted molecules among all bone and marrow cells including myeloma cells. Therefore, to explore the mechanism underlying osteocyte apoptosis induced by myeloma cells we used a co-culture system that allows both cell-to-cell contact and exchange of soluble factors between osteocytic MLO-A5 cells and the human JJN3 MM cell line or primary CD138+ cells isolated from MM patients. Osteocyte apoptosis was quantified by trypan blue uptake and chromatin condensation/nuclear fragmentation in the absence or presence of the caspase3 specific inhibitor DEVD. We found that osteocyte apoptosis was increased 2-3 fold when osteocytes were co-cultured with JJN3 cells compared to osteocytes cultured alone. Apoptosis occurred within 8h of co-culture and gradually increased for up to 48h. Osteocyte apoptosis was completely inhibited by DEVD. Importantly, co-culture with primary CD138+ MM cells from 5 different patients also increased osteocyte apoptosis, which was also blocked by DEVD. We next determined if direct cell-to-cell contact was required for MM cells to induce osteocyte apoptosis and whether Notch signaling, a signaling pathway with profound effects on the skeleton activated by such interactions, was involved. We found that osteocytes co-cultured with JJN3 cells exhibited 3-7 fold higher levels of expression of the Notch target genes Hes1 and Hey1, detected as early as 4h and maintained up to 48h of co-culture. The pharmacological specific Notch inhibitor GSIXX completely blocked osteocyte apoptosis induced by either JJN3 cells or primary CD138+ MM cells measured at 8h and 24h. Moreover, cultures of osteocytes grown on plates coated with the Notch ligand Delta 1 fused to IgG2 exhibited 2-5 times higher levels of apoptosis compared to osteocytes cultured on IgG2 control, and this effect was inhibited by GSIXX. In addition, overexpression of the Notch intracellular domains 1 or 2, known to activate Notch signaling, increased osteocyte apoptosis by 2 fold. These findings demonstrate that rapid activation Notch signaling in osteocytes triggered by direct cell-to-cell contact with myeloma cells induces osteocyte apoptosis. Interestingly, Notch inhibition by GSIXX only partially prevented osteocyte apoptosis induced by JJN3 cells measured at 48h, suggesting the involvement of other mechanisms. To further investigate this finding, we measured soluble levels of TNFα in JJN3 cultures since TNFα is a recognized inducer of osteocyte apoptosis. The levels of TNFα secreted by JJN3 cells increased 5 fold (from 2 to 9 pg/ml) during 4 to 48h of culture. Conditioned medium (CM) from JJN3 cells cultured alone for 48h increased osteocyte apoptosis, and this effect was blocked by DEVD and by a neutralizing anti-human TNFα antibody, but not by GSIXX. Moreover, combination of GSIXX and anti-TNFα antibody completely inhibited osteocyte apoptosis induced by co-culture with JJN3 cells, while each agent added separately only partially inhibited osteocyte apoptosis measured at 48h. These results demonstrate that direct interactions with MM cells induces caspase3-dependent osteocyte apoptosis, triggered by rapid activation of Notch signaling through cell-cell contact and is maintained by accumulation of MM-derived TNFα. Our findings suggest both Notch and TNFα signaling pathways are potential targets to reverse or prevent myeloma induced osteocyte apoptosis. DisclosuresNo relevant conflicts of interest to declare.

IL-17A-Mediated Notch Signaling in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy, however, significant abnormalities in T cell function are considered to provide help in uncontrolled growth and survival of MM cells. We have previously reported that IL-17A-producing Th17 cells are elevated in MM, that MM cells express IL-17 receptor, and IL-17A promotes MM cell growth and survival. We have reported that MM cells themselves produce IL-17A as confirmed by RT-PCR, Western blotting and immunostaining providing a possibility of both autocrine and paracrine role for IL-17A in MM. As Notch activation has been implicated in Th17 cell differentiation and IL17A production, we have here investigated the role of Notch pathway activation in IL-17A-mediated MM cell growth within the BM microenvironment. Notch consists of 4 proteins (1-4) and has 5 ligands (DLL-1,3,4 and jagged-1, 2). We analyzed RNA-Seq data from 117 newly-diagnosed MM patients and 18 normal plasma cells and observed high expression of Notch 1, and 2 and Notch target genes Hes-1 and Hey-1 but not Notch 3 and 4 in MM. For Notch 2, isoform 2 was highly predominant. Notch expression on MM cells was further confirmed by flow cytometric analysis (Notch1-84%, Notch2-86% and Notch3-3%). Evaluating functional role of Notch in MM, when MM cells were co-cultured with Notch ligand jagged 2-expressing 3T3 cells, IL-17A was able to further induce Notch target gene Hes-1 by 45%. Interestingly, increase in the expression of Notch 2 was also observed during this interaction (increased full-length protein by 65% and active intra-cellular protein by 145%). We next evaluated effect of both anti-IL-17 antibody and Notch inhibitors on MM cells. Anti-IL-17A monoclonal antibody inhibited full-length Notch2 protein expression by 54% and active intra-cellular protein by 85%, as determined by western blot analysis. The antibody inhibitory activity was confirmed with quantitative PCR. Importantly, IL-17A mAb inhibited Hes-1 protein expression by 83%. With the observed impact of Notch signaling in MM, we next evaluated notch inhibitors MRK003, and compound E, a γ-secretase inhibitors, to determine their impact on MM cell growth and survival. We observe that Notch inhibitors affect MM cell growth (inhibition by 43%%, N=5) and IL-6 production (inhibition by 60%, N=3) in co-culture with bone marrow stromal cells. These preclinical data establish the role of IL-17 as well as Notch signaling in myeloma and provides the rationale to evaluate anti-MM activity of anti-IL-17A monoclonal antibody and Notch inhibitors in MM. DisclosuresNo relevant conflicts of interest to declare.

Connective Tissue Growth Factor is a Target of Notch Signaling in Cells of the Osteoblastic Lineage

Connective tissue growth factor (Ctgf) or CCN2 is a protein synthesized by osteoblasts necessary for skeletal homeostasis, although its overexpression inhibits osteogenic signals and bone formation. Ctgf is induced by bone morphogenetic proteins, transforming growth factor β and Wnt; and in the present studies, we explored whether Notch regulated Ctgf expression in osteoblasts. We employed RosaNotch mice, where the Notch intracellular domain (NICD) is expressed following the excision of a STOP cassette, placed between the Rosa26 promoter and NICD. Notch was activated by transduction of adenoviral vectors expressing Cre recombinase (Ad-CMV-Cre). Notch induced Ctgf mRNA levels in a time dependent manner and increased Ctgf heterogeneous nuclear RNA. Notch also destabilized Ctgf mRNA shortening its half-life from 13h to 3h. The effect of Notch on Ctgf expression was lost following Rbpjκ downregulation, demonstrating that it was mediated by Notch canonical signaling. However, downregulation of the classic Notch target genes Hes1, Hey1 and Hey2 did not modify the effect of Notch on Ctgf expression. Wild type osteoblasts exposed to immobilized Delta-like 1 displayed enhanced Notch signaling and increased Ctgf expression. In addition to the effects of Notch in vitro, Notch induced Ctgf in vivo, and calvariae and femurs from RosaNotch mice mated with transgenics expressing the Cre recombinase in cells of the osteoblastic lineage exhibited increased expression of Ctgf. In conclusion, Ctgf is a target of Notch canonical signaling in osteoblasts, and may act in concert with Notch to regulate skeletal homeostasis.

Prostate tumor OVerexpressed-1 (PTOV1) down-regulates HES1 and HEY1 notch targets genes and promotes prostate cancer progression

BackgroundPTOV1 is an adaptor protein with functions in diverse processes, including gene transcription and protein translation, whose overexpression is associated with a higher proliferation index and tumor grade in prostate cancer (PC) and other neoplasms. Here we report its interaction with the Notch pathway and its involvement in PC progression.MethodsStable PTOV1 knockdown or overexpression were performed by lentiviral transduction. Protein interactions were analyzed by co-immunoprecipitation, pull-down and/or immunofluorescence. Endogenous gene expression was analyzed by real time RT-PCR and/or Western blotting. Exogenous promoter activities were studied by luciferase assays. Gene promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP). In vivo studies were performed in the Drosophila melanogaster wing, the SCID-Beige mouse model, and human prostate cancer tissues and metastasis. The Excel package was used for statistical analysis.ResultsKnockdown of PTOV1 in prostate epithelial cells and HaCaT skin keratinocytes caused the upregulation, and overexpression of PTOV1 the downregulation, of the Notch target genes HEY1 and HES1, suggesting that PTOV1 counteracts Notch signaling. Under conditions of inactive Notch signaling, endogenous PTOV1 associated with the HEY1 and HES1 promoters, together with components of the Notch repressor complex. Conversely, expression of active Notch1 provoked the dismissal of PTOV1 from these promoters. The antagonist role of PTOV1 on Notch activity was corroborated in the Drosophila melanogaster wing, where human PTOV1 exacerbated Notch deletion mutant phenotypes and suppressed the effects of constitutively active Notch. PTOV1 was required for optimal in vitro invasiveness and anchorage-independent growth of PC-3 cells, activities counteracted by Notch, and for their efficient growth and metastatic spread in vivo. In prostate tumors, the overexpression of PTOV1 was associated with decreased expression of HEY1 and HES1, and this correlation was significant in metastatic lesions.ConclusionsHigh levels of the adaptor protein PTOV1 counteract the transcriptional activity of Notch. Our evidences link the pro-oncogenic and pro-metastatic effects of PTOV1 in prostate cancer to its inhibitory activity on Notch signaling and are supportive of a tumor suppressor role of Notch in prostate cancer progression.

Mouse Sertoli Cells Sustain De Novo Generation of Regulatory T Cells by Triggering the Notch Pathway Through Soluble JAGGED11

FOXP3(+) regulatory T cells (Tregs) are central to the maintenance of immunological homeostasis and tolerance. It has long been known that Sertoli cells are endowed with immune suppressive properties; however, the underlying mechanisms as well as the effective nature and role of soluble factors secreted by Sertoli cells have not been fully elucidated as yet. We hypothesized that conditioned medium from primary mouse Sertoli cells (SCCM) may be able and sufficient to induce Tregs. By culturing CD4(+)CD25(-)EGFP(-) T splenocytes purified from FOXP3-EGFP knock-in mice in SCCM, here we show, by flow cytometry and suppression assay, the conversion of peripheral CD4(+)FOXP3(-) T cells into functional CD4(+)FOXP3(+) Tregs. We also demonstrate that the Notch/Jagged1 axis is involved in regulating the de novo generation of Tregs although this process is transforming growth factor-beta1 (TGF-B) dependent. In particular, we identified by Western blot analysis a soluble form of JAGGED1 (JAG1) in SCCM that significantly influences the induction of Tregs, as demonstrated by performing the conversion assay in presence of a JAG1-specific neutralizing antibody. In addition, we show that SCCM modulates the Notch pathway in converted Tregs by triggering the recruitment of the Notch-specific transcription factor CSL/RBP-Jk to the Foxp3 promoter and by inducing the Notch target gene Hey1, as shown by chromatin immunoprecipitation assay and by real time-RT-PCR experiments, respectively. Overall, these results contribute to a better understanding of the molecular mechanisms involved in Sertoli cell-mediated immune tolerance and provide a novel approach to generate ex vivo functional Tregs for therapeutic purpose.

The 10 Most Read Articles Published in Circulation Research in 2013

The 10 Most Read Articles Published in <i>Circulation Research</i> in 2013

Regulation of endothelial signaling and migration by v-ATPase

The vacuolar ATPase (v-ATPase) is a proton pump, able to acidify intracellular compartments and the pericellular space. v-ATPase has extensively been studied in various functional contexts, e.g., migration of tumor cells, and inhibition of v-ATPase has been proven as intriguing novel therapeutic concept. Since the role of v-ATPase in endothelial cell migration and angiogenesis has scarcely been investigated, we examined the consequences of pharmacological inhibition of v-ATPase (by concanamycin) on proliferation, migration, VEGF-receptor 2 (VEGFR2) trafficking and signaling, as well as Notch-mediated transcription in endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC)] Treatment of the cells with 3 or 10 nM of the v-ATPase inhibitor concanamycin for 48 h or longer inhibited proliferation and arrested cell cycle in the G2/M phase in HMEC-1, while a G1 phase arrest occurred in HUVEC. Already after 24 h these concentrations reduced migration (scratch assay, chemotactic gradient). Activation of the small GTPase Rac1 in freshly adherent cells was reduced by concanamycin. Downstream signaling of the VEGFR2 (phosphorylation of ERK1/2 and AKT), as well as autophosphorylation of VEGFR2 were inhibited. VEGFR2 on the cell surface was reduced, and sequestered in a lysosomal compartment. In addition, concanamycin blocked transcription of the Notch target genes Hey1 and Hey2 after stimulation with DLL4. Since the impaired signaling pathways (Rac-1, VEGFR2, Notch) all depend on vesicular recycling circuits, we conclude that the disturbance of these is the main mode of action of v-ATPase inhibition in endothelial cells, offering an attractive multi-factorial anti-angiogenic approach.

Synaptojanin-2 Binding Protein Stabilizes the Notch Ligands DLL1 and DLL4 and Inhibits Sprouting Angiogenesis

The formation of novel blood vessels is initiated by vascular endothelial growth factor. Subsequently, DLL4-Notch signaling controls the selection of tip cells, which guide new sprouts, and trailing stalk cells. Notch signaling in stalk cells is induced by DLL4 on the tip cells. Moreover, DLL4 and DLL1 are expressed in the stalk cell plexus to maintain Notch signaling. Notch loss-of-function causes formation of a hyperdense vascular network with disturbed blood flow. This study was aimed at identifying novel modifiers of Notch signaling that interact with the intracellular domains of DLL1 and DLL4. Synaptojanin-2 binding protein (SYNJ2BP, also known as ARIP2) interacted with the PDZ binding motif of DLL1 and DLL4, but not with the Notch ligand Jagged-1. SYNJ2BP was preferentially expressed in stalk cells, enhanced DLL1 and DLL4 protein stability, and promoted Notch signaling in endothelial cells. SYNJ2BP induced expression of the Notch target genes HEY1, lunatic fringe (LFNG), and ephrin-B2, reduced phosphorylation of ERK1/2, and decreased expression of the angiogenic factor vascular endothelial growth factor (VEGF)-C. It inhibited the expression of genes enriched in tip cells, such as angiopoietin-2, ESM1, and Apelin, and impaired tip cell formation. SYNJ2BP inhibited endothelial cell migration, proliferation, and VEGF-induced angiogenesis. This could be rescued by blockade of Notch signaling or application of angiopoietin-2. SYNJ2BP-silenced human endothelial cells formed a functional vascular network in immunocompromised mice with significantly increased vascular density. These data identify SYNJ2BP as a novel inhibitor of tip cell formation, executing its functions predominately by promoting Delta-Notch signaling.

Abstract 4762: Anti-Nicastrin antibodies for the treatment of endocrine resistant breast cancer .

Abstract Background: Resistance to endocrine therapy occurs in at least 30% of patients with breast cancer (BC) and remains a major clinical problem. Among the mechanisms underpinning endocrine resistance is the enrichment of epithelial-to-mesenchymal transition (EMT) features and signalling pathways such as EGFR and Notch. Notch receptors are activated by the gamma-secretase (GS) enzyme composed of nicastrin, presenilin, PEN2 and Aph-1. Gamma secretase inhibitors are being tested as novel therapeutics for cancer treatment, predominantly in combination with anti-hormonal agents as well as chemotherapy, however treatment related toxicities are apparent. Nicastrin is upregulated in BC and we have developed anti-nicastrin monoclonal antibodies (MAbs) to target its functions and block GS. Here, we show that nicastrin may be a relevant therapeutic target in endocrine resistance and demonstrate the effects of anti-nicastrin MAbs. Materials and Methods: We used two cell line models of MCF7 cells which developed endocrine resistance: TamR cells (Hiscox et al) and MLET2/MLET5 developed in our laboratory. We raised anti-nicastrin MAbs (MAb1 and MAb2) against nicastrin extracellular domain. Western blotting, RT-qPCR, transwell invasion and 3D matrigel growth assays were used to demonstrate their molecular and functional effects. Gamma secreatse inhibitor RO4929097 was used for comparison. Results: We demonstrate that nicastrin is overexpressed at both mRNA and protein level in endocrine resistant cell lines (&amp;gt; 4-fold). These cells concomitantly exhibit elevated molecular markers of EMT: Vimentin (3-fold), CD44 (&amp;gt; 2-fold), EGFR (&amp;gt; 4-fold), RhoGTPase Cdc42 target genes IQGAP1 (2-fold) and IQGAP2 (&amp;gt;100-fold); as well as Notch4 (&amp;gt;2-fold), Notch-targets Hey1 and Hes1 (4-fold), while Notch1 is suppressed (&amp;gt; 2-fold). Anti-nicastrin MAb1, MAb2 and RO4929097 were equally potent in inhibiting Notch target genes Hey1 and Hes1, as well as growth of TAMR and MLET acini in 3D Matrigel. Interestingly, effective inhibition of vimentin, CD44 and IQGAPs was observed only upon treatment with anti-nicastrin MAbs, while RO4929097 had no effect. Interestinly, a clear distinction was noted between anti-nicastrin MAbs and RO4929097 in terms of their effect on Notch4. MAb1 and MAb2 attenuated Notch4 transcription and reduced activated Notch4 and nicastrin levels, while RO4929097 achieved an opposite effect. Furthermore, only anti-nicastrin MAbs were also potent inhibitors of TAMR cell invasion (by &amp;gt;50%, p&amp;lt;0.01). Conclusion: Nicastrin is overexpressed in endocrine resistant breast cancer cells. Anti-nicastrin monoclonal antibodies inhibit Notch4 signalling, attenuate expression of pro-invasive molecules such as vimentin, IQGAPs and CD44 and reduce cellular invasiveness and growth in 3D Matrigel. Therefore, targeting nicastrin may be a valid therapeutic strategy in breast cancer patients that relapse of endocrine therapy. Citation Format: Monica Faronato, Ylenia Lombardo, Aleksandra Filipovic, Simak Ali, R.Charles Coombes. Anti-Nicastrin antibodies for the treatment of endocrine resistant breast cancer . [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 4762. doi:10.1158/1538-7445.AM2013-4762

Notch Transcriptional Control of Vascular Smooth Muscle Regulatory Gene Expression and Function

Notch receptors and ligands mediate heterotypic cell signaling that is required for normal vascular development. Dysregulation of select Notch receptors in mouse vascular smooth muscle (VSM) and in genetic human syndromes causes functional impairment in some regional circulations, the mechanistic basis of which is undefined. In this study, we used a dominant-negative Mastermind-like (DNMAML1) to block signaling through all Notch receptors specifically in VSM to more broadly test a functional role for this pathway in vivo. Mutant DNMAML1-expressing mice exhibited blunted blood pressure responses to vasoconstrictors, and their aortic, femoral, and mesenteric arteries had reduced contractile responses to agonists and depolarization in vitro. The mutant arteries had significant and specific reduction in the expression and activity of myosin light chain kinase (MLCK), a primary regulator of VSM force production. Conversely, activated Notch signaling in VSM cells induced endogenous MLCK transcript levels. We identified MLCK as a direct target of activated Notch receptor as demonstrated by an evolutionarily conserved Notch-responsive element within the MLCK promoter that binds the Notch receptor complex and is required for transcriptional activity. We conclude that Notch signaling through the transcriptional control of key regulatory proteins is required for contractile responses of mature VSM. Genetic or pharmacological manipulation of Notch signaling is a potential strategy for modulating arterial function in human disease.

COUP-TFII orchestrates venous and lymphatic endothelial identity by homo- or hetero-dimerisation with PROX1

Endothelial cell (EC) identity is in part genetically predetermined. Transcription factor NR2F2 (also known as chicken ovalbumin upstream promoter transcription factor II, COUP-TFII) plays a key role in EC fate decision making; however, many of the underlying mechanisms remain enigmatic. In the present study, we demonstrate that NR2F2 differentially regulates gene expression of venous versus lymphatic ECs (LECs) and document a novel paradigm whereby NR2F2 homodimers induce a venous EC fate, while heterodimers with the LEC-specific transcription factor PROX1 instruct LEC lineage specification. NR2F2 homodimers inhibit arterial differentiation in venous ECs through direct binding to the promoter regions of the Notch target genes HEY1 and HEY2 (HEY1/2), whereas NR2F2/PROX1 heterodimers lack this inhibitory effect, resulting at least in part in non-canonical HEY1/2 expression in LECs. Furthermore, NR2F2/PROX1 heterodimers actively induce or are permissive for the expression of a major subset of LEC-specific genes. In addition to NR2F2/PROX1 heterodimerisation, the expression of HEY1 and some of these LEC-specific genes is dependent on PROX1 DNA binding. Thus, NR2F2 homodimers in venous ECs and NR2F2/PROX1 heterodimers in LECs differentially regulate EC subtype-specific genes and pathways, most prominently the Notch target genes HEY1/2. This novel mechanistic insight could pave the way for new therapeutic interventions for vascular-bed-specific disorders.

Crosstalk between the canonical NF-κB and Notch signaling pathways inhibits Pparγ expression and promotes pancreatic cancer progression in mice

The majority of human pancreatic cancers have activating mutations in the KRAS proto-oncogene. These mutations result in increased activity of the NF-κB pathway and the subsequent constitutive production of proinflammatory cytokines. Here, we show that inhibitor of κB kinase 2 (Ikk2), a component of the canonical NF-κB signaling pathway, synergizes with basal Notch signaling to upregulate transcription of primary Notch target genes, resulting in suppression of antiinflammatory protein expression and promotion of pancreatic carcinogenesis in mice. We found that in the Kras(G12D)Pdx1-cre mouse model of pancreatic cancer, genetic deletion of Ikk2 in initiated pre-malignant epithelial cells substantially delayed pancreatic oncogenesis and resulted in downregulation of the classical Notch target genes Hes1 and Hey1. Tnf-α stimulated canonical NF-κB signaling and, in collaboration with basal Notch signals, induced optimal expression of Notch targets. Mechanistically, Tnf-α stimulation resulted in phosphorylation of histone H3 at the Hes1 promoter, and this signal was lost with Ikk2 deletion. Hes1 suppresses expression of Pparg, which encodes the antiinflammatory nuclear receptor Pparγ. Thus, crosstalk between Tnf-α/Ikk2 and Notch sustains the intrinsic inflammatory profile of transformed cells. These findings reveal what we believe to be a novel interaction between oncogenic inflammation and a major cell fate pathway and show how these pathways can cooperate to promote cancer progression.

Silk peptides inhibit adipocyte differentiation through modulation of the Notch pathway in C3H10T1/2 cells

Silk protein is a biocompatible material that has been used in many biotechnological applications and exhibits body fat-lowering effects. Recent studies have shown that silk peptides increase expression of osteogenic markers in osteoblast-like cells. Because osteogenic and adipogenic differentiation from common mesenchymal progenitor cells are inverse processes and often regulated reciprocally, we hypothesized that silk peptides might suppress adipocyte differentiation. We therefore endeavored to evaluate the effects of silk peptides on adipocyte differentiation in C3H10T1/2 cells. We find that silk peptides inhibit lipid accumulation and morphological differentiation in these cells. Molecular studies show that silk peptides block expression of adipocyte-specific genes such as peroxisome proliferator-activated receptor γ and its targets, including aP2, Cd36, CCAAT enhancer binding proteinα. Silk peptides appear to inhibit adipogenesis by suppression of the Notch pathway, repressing the Notch target genes Hes-1 and Hey-1. In addition, these peptides inhibit endogenous Notch activation, as shown by a reduction in generation of Notch intracellular domain. N-[N-(3.5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester, compound E, and WPE-III-31C, which are all known Notch signaling inhibitors, block adipocyte differentiation to an extent similar to silk peptides. Together, our data demonstrate that silk peptides can modulate adipocyte differentiation through inhibition of the Notch signaling and further suggest potential future strategies for treating obesity and its related metabolic diseases.

Notch Initiates the Endothelial-to-Mesenchymal Transition in the Atrioventricular Canal through Autocrine Activation of Soluble Guanylyl Cyclase

The heart is the most common site of congenital defects, and valvuloseptal defects are the most common of the cardiac anomalies seen in the newborn. The process of endothelial-to-mesenchymal transition (EndMT) in the cardiac cushions is a required step during early valve development, and Notch signaling is required for this process. Here we show that Notch activation induces the transcription of both subunits of the soluble guanylyl cyclase (sGC) heterodimer, GUCY1A3 and GUCY1B3, which form the nitric oxide receptor. In parallel, Notch also promotes nitric oxide (NO) production by inducing Activin A, thereby activating a PI3-kinase/Akt pathway to phosphorylate eNOS. We thus show that the activation of sGC by NO through a Notch-dependent autocrine loop is necessary to drive early EndMT in the developing atrioventricular canal (AVC).

A SIRT1-LSD1 Corepressor Complex Regulates Notch Target Gene Expression and Development

Epigenetic regulation of gene expression by histone-modifying corepressor complexes is central to normal animal development. The NAD(+)-dependent deacetylase and gene repressor SIRT1 removes histone H4K16 acetylation marks and facilitates heterochromatin formation. However, the mechanistic contribution of SIRT1 to epigenetic regulation at euchromatic loci and whether it acts in concert with other chromatin-modifying activities to control developmental gene expression programs remain unclear. We describe here a SIRT1 corepressor complex containing the histone H3K4 demethylase LSD1/KDM1A and several other LSD1-associated proteins. SIRT1 and LSD1 interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress genes regulated by the Notch signaling pathway. Mutations in Drosophila SIRT1 and LSD1 orthologs result in similar developmental phenotypes and genetically interact with the Notch pathway in Drosophila. These findings offer new insights into conserved mechanisms of epigenetic gene repression and regulation of development by SIRT1 in metazoans.