Active Notch Intracellular Domain Research Articles

Dangerous Liaisons: Deviant Endothelium NOTCHes toward Tumor Metastasis

In this issue of Cancer Cell, Wieland etal. uncover a feedback loop in which tumor cells, by augmenting Notch signaling, provoke a senescent and pro-inflammatory state in endothelial cells, promoting neutrophil infiltration, tumor cell adhesion, and metastasis. Interfering with this Notch-dependent crosstalk may be a therapeutic approach to block metastasis.

Suberoyl bis-hydroxamic acid activates Notch1 signaling and induces apoptosis in anaplastic thyroid carcinoma through p53.

Anaplastic thyroid cancer (ATC), usually derived from well-differentiated thyroid cancers is one of the most lethal human endocrine malignancies. In the present study, we report that in human ATC tumor tissue samples exist undetectable Notch1 and the active Notch1 intracellular domain could not be detected in ATC-CAL-62 cells. Interesting, suberoyl bis-hydroxamic acid (SBHA) administration could induce Notch1 intracellular domain levels in a dose-dependent manner, coupled with the increase of p53 and p21. Furthermore, ectopic expression of Notch1 or deletion of p53 with small-interfering RNA was able to abolish the effects of SBHA to elevation of Notch1 and p53 in ATC cells. As a result, SBHA treatment efficiently induced ATC cell apoptosis. These results indicate that SBHA may play antitumor functions via regulating Notch1/p53 signals, and highlight that SBHA could have clinical potential to benefit the therapy of ATC patients.

Altered DNA Methylation of Long Noncoding RNA H19 in Calcific Aortic Valve Disease Promotes Mineralization by Silencing NOTCH1.

Calcific aortic valve disease is characterized by an abnormal mineralization of the aortic valve. Osteogenic activity in the aortic valve is under the control of NOTCH1, which regulates the expression of key pro-osteogenic genes such as RUNX2 and BMP2. Long noncoding RNAs (lncRNAs) may reprogram cells by altering the gene expression pattern. Multidimensional genomic profiling was performed in human aortic valves to document the expression of lncRNAs and the DNA methylation pattern in calcific aortic valve disease. In-depth functional assays were carried out to document the impact of lncRNA on the mineralization of the aortic valve. We documented that lncRNA H19 (H19) was increased in calcific aortic valve disease. Hypomethylation of the promoter region was observed in mineralized aortic valves and was inversely associated with H19 expression. Knockdown and overexpression experiments showed that H19 induces a strong osteogenic phenotype by altering the NOTCH1 pathway. Gene promoter analyses showed that H19 silenced NOTCH1 by preventing the recruitment of p53 to its promoter. A knockdown of H19 in valve interstitial cells (VICs) increased the expression of NOTCH1 and decreased the level of RUNX2 and BMP2, 2 downstream targets repressed by NOTCH1. In rescue experiments, the transfection of a vector encoding for the active Notch intracellular domain prevented H19-induced mineralization of valve interstitial cells. These findings indicate that a dysregulation of DNA methylation in the promoter of H19 during calcific aortic valve disease is associated with a higher expression of this lncRNA, which promotes an osteogenic program by interfering with the expression of NOTCH1.

The Tetraspanin TSPAN33 Controls TLR-Triggered Macrophage Activation through Modulation of NOTCH Signaling.

The involvement of NOTCH signaling in macrophage activation by Toll receptors has been clearly established, but the factors and pathways controlling NOTCH signaling during this process have not been completely delineated yet. We have characterized the role of TSPAN33, a tetraspanin implicated in a disintegrin and metalloproteinase (ADAM) 10 maturation, during macrophage proinflammatory activation. Tspan33 expression increases in response to TLR signaling, including responses triggered by TLR4, TLR3, and TLR2 activation, and it is enhanced by IFN-γ. In this study, we report that induction of Tspan33 expression by TLR and IFN-γ is largely dependent on NOTCH signaling, as its expression is clearly diminished in macrophages lacking Notch1 and Notch2 expression, but it is enhanced after overexpression of a constitutively active intracellular domain of NOTCH1. TSPAN33 is the member of the TspanC8 tetraspanin subgroup more intensely induced during macrophage activation, and its overexpression increases ADAM10, but not ADAM17, maturation. TSPAN33 favors NOTCH processing at the membrane by modulating ADAM10 and/or Presenilin1 activity, thus increasing NOTCH signaling in activated macrophages. Moreover, TSPAN33 modulates TLR-induced proinflammatory gene expression, at least in part, by increasing NF-κB-dependent transcriptional activity. Our results suggest that TSPAN33 represents a new control element in the development of inflammation by macrophages that could constitute a potential therapeutic target.

Inhibition of γ-secretase activity synergistically enhances tumour necrosis factor-related apoptosis-inducing ligand induced apoptosis in T-cell acute lymphoblastic leukemia cells via upregulation of death receptor 5.

T-cell acute lymphoblastic leukemia (T-ALL) is a rare and aggressive hematopoietic malignancy prone to relapse and drug resistance. Half of all T-ALL patients exhibit mutations in Notch1, which leads to aberrant Notch1 associated signaling cascades. Notch1 activation is mediated by the γ-secretase cleavage of the Notch1 receptor into the active intracellular domain of Notch1 (NCID). Clinical trials of γ-secretase small molecule inhibitors (GSIs) as single agents for the treatment of T-ALL have been unsuccessful. The present study demonstrated, using immunofluorescence and western blotting, that blocking γ-secretase activity in T-ALL cells with N-[(3,5-difluorophenyl) acetyl]-L-alanyl-2-phenyl] glycine-1,1-dimethylethyl ester (DAPT) downregulated NCID and upregulated the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5). Upregulation of DR5 restored the sensitivity of T-ALL cells to TRAIL. Combination index revealed that the combined treatment of DAPT and TRAIL synergistically enhanced apoptosis compared with treatment with either drug alone. TRAIL combined with the clinically evaluated γ-secretase inhibitor 3-[(1r, 4s)-4-(4-chlorophenylsulfonyl)-4-(2, 5-difluorophenyl) cyclohexyl] propanoic acid (MK-0752) also significantly enhanced TRAIL-induced cell death compared with either drug alone. DAPT/TRAIL apoptotic synergy was dependent on the extrinsic apoptotic pathway and was associated with a decrease in BH3 interacting-domain death agonist and x-linked inhibitor of apoptosis. In conclusion, γ-secretase inhibition represents a potential therapeutic strategy to overcome TRAIL resistance for the treatment of T-ALL.

Abstract 386: Notch1 Signaling Negatively Modulates Repolarizing Kv Currents in Cardiomyocytes

Notch1 is a critical signaling pathway during embryonic and early postnatal development and activity of this transduction system gradually declines after birth. Conversely, outward K + Kv currents are progressively increased in myocytes during postnatal life leading to shortening of the action potential (AP) duration and acquisition of the mature electrical phenotype. Thus, we tested the possibility that Notch1 signaling modulates the electrophysiological properties of developing cells by interfering with Kv current densities. For this purpose, molecular and physiological studies were conducted in vitro using mouse neonatal myocytes (NMs). These assays were complemented with tests in adult cells obtained from a conditional mouse model of Notch1 intracellular domain (NICD) overexpression. NMs were treated with a γ-secretase inhibitor (DAPT) to prevent the cleavage of Notch1 receptor and subsequent translocation of the active NICD to the nucleus. By RT-PCR, Notch1 targets Hes1 and Hey1 were reduced with DAPT-treatment, confirming the effective perturbation of Notch1 signaling. Importantly, inhibition of Notch signaling led to upregulation of the three splice variants of the Kv channel-interacting proteins 2 (KChIP2) gene, which controls the appearance of outward Kv currents in myocytes. By patch-clamp, Kv currents were identified in only 15% of NMs (n=20) cultured for 1-2 days in in control condition, whereas this fraction increased to 50% (n=18) in cells treated with DAPT. Thus, Notch inhibition promotes the expression of KChIP2 and appearance of Kv currents. To clarify these in vitro findings, the consequences of ectopic activation of Notch1 signaling on the electrical behavior of adult myocytes were established. By patch-clamp, NICD-overexpressing myocytes presented a >2-fold longer duration of the early repolarization phase of the AP, with respect to cells from wild type hearts. This alteration was coupled with a >50% reduction of Kv currents I to and I Kslow1 in NICD-overexpressing cells, with respect to wild-type myocytes. Thus, Notch signaling represses KChIP2 and Kv currents in cardiomyocytes representing an important modulator of the electrical phenotype of the developing heart.

Abstract 1133: Prevalence of activated NOTCH receptor in solid tumors and chronic lymphocytic leukemia

Abstract Recent reports identified high expression of Notch 1 receptor in various tumors including adenoid cystic carcinomas (ACC). Notch 1 receptor is activated by gamma secretase cleavage to exert transcriptional control of processes including cellular differentiation and proliferation. Notch is also known to be activated in approximately 60% of T-cell acute lymphoblastic leukemias. We investigated the prevalence of Notch 1, 2 and 3 activation in different tumors by immunohistochemistry (IHC) designed separately for Notch 1, 2 and 3. Our proprietary antibodies recognize the post-gamma secretase cleavage activated Notch intracellular domain (NICD) fragments of Notch 1, Notch 2 and Notch 3, allowing direct measurement of activation of these receptors, in addition to their expression level by the tumor cells. In total we tested 48 ACCs of the salivary glands, 19 breast cancer, 35 chronic lymphocytic leukemias (CLL), 7 glioblastomas, and 35 non-small cell lung carcinomas (NSCLC) We found that at least 65% (31 out of 48) of ACCs were positive for Notch 1 activation using our assay. Of other tumor types, 4 out of 7 glioblastomas (57%), 14 out of 35 CLLs (40%), 7 out of 35 NSCLC (20%) and 2 out of 19 breast cancer cases (11%) were positive for Notch 1. Within NSCLC, 5 of the 16 tested squamous cell carcinomas were positive for Notch 1 activation (31%). Notch 2 and Notch 3 showed only rare activation in these tumors. We conclude that high levels of Notch 1 activation are identifiable in different tumor types including high prevalence in ACC, CLL and glioma. These tumors may benefit from therapeutic Notch pathway inhibition. Citation Format: Gerard Oakley, Lisa Riegle, Karim Benhadji, Andrew Schade. Prevalence of activated NOTCH receptor in solid tumors and chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1133.

Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival.

ABSTRACTEhrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival.

Notch Signaling Rescues Loss of Satellite Cells Lacking Pax7 and Promotes Brown Adipogenic Differentiation.

Pax7 is a nodal transcription factor that is essential for regulating the maintenance, expansion, and myogenic identity of satellite cells during both neonatal and adult myogenesis. Deletion of Pax7 results in loss of satellite cells and impaired muscleregeneration. Here, we show that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) rescues the loss of Pax7-deficient satellite cells and restores their proliferative potential. Strikingly NICD1-expressing satellite cells do not undergo myogenic differentiation and instead acquire a brown adipogenic fate both invivo and invitro. NICD-expressing Pax7(-/-) satellite cells fail to upregulate MyoD and instead express the brown adipogenic marker PRDM16. Overall, these results show that Notch1 activation compensates for the loss of Pax7 in the quiescent state and acts as a molecular switch topromote brown adipogenesis in adult skeletal muscle.

PTU-085 A NOTCH1-Driven Secretome Switch Modulates Immune Surveillance In Ras-Induced Senescence

Introduction Oncogene-induced senescence (OIS) is an intrinsic tumour suppressor mechanism, but its impact on tumorigenesis is largely dependent on the nature of the senescence-associated secretory phenotype (SASP). Major components of the SASP include TGFβ1 and pro-inflammatory cytokines, such as IL1, IL6, and IL8, that have pleiotropic context-dependent effects. Methods We utilised the well validated ER:Ras G12V IMR90 HDF in vitro model which undergo Ras-induced senescence (RIS) with 4 OHT. Genetic manipulation was achieved through retroviral gene transfer; transcriptional profiling by mRNA-seq; validation through qPCR and immunoblotting. In vivo hepatocyte senescence was achieved through hydrodynamic tail-vein delivery of NRAS G12V -containing transposons. Results We have demonstrated that NOTCH1, a highly conserved receptor is up-regulated in RIS. In contrast to the up-regulation of NOTCH1, downstream signalling is dynamically regulated: the cleaved, active intracellular domain of NOTCH1 (N1ICD) and NOTCH-target genes were transiently up-regulated at an early phase of RIS, but down-regulated at full senescence. The dynamic expression patterns of N1ICD and TGF-β1 expression were nearly identical, and inversely correlated with the cytokines, IL1 and IL8. Inhibition of NOTCH1 signalling, through expression of a dominant-negative form of the NOTCH1 binding partner MAML1, led to a reduction in TGF-β1, but increased IL1 and IL8 expression during RIS. In addition, ectopic restoration of N1ICD in established RIS cells drove reciprocal secretome changes with reduced IL1 and IL8 and increased TGF-β1, suggesting NOTCH1 signalling plays a critical role in secretome switching. Utilising an NRAS-drive hepatocyte senescence model, NOTCH1 was upregulated during RIS in vivo. Previously it has been shown that the SASP in RIS is critical for the immune-mediated clearance of senescent hepatocytes. Consistent with the enhancement of the pro-inflammatory secretome, co-delivery of the NOTCH inhibitor dnMAML1 with RAS, accelerated the infiltration of CD3+ T-cells into the liver and promoted the clearance of senescent hepatocytes. Restoration of N1ICD in hepatocyte RIS led to senescence bypass and tumour formation. Conclusion The transition to RIS is correlated with a NOTCH1-mediated switch from a TGFβ-rich secretome to a proinflammatory secretome. Promotion of this proinflammatory secretome, through NOTCH inhibition enhances immune surveillance of senescent hepatocytes. This raises the possibility of therapeutically promoting the clearance of senescent cells in vivo. Disclosure of Interest None Declared

Inhibition of endothelial Cdk5 reduces tumor growth by promoting non-productive angiogenesis.

Therapeutic success of VEGF-based anti-angiogenic tumor therapy is limited due to resistance. Thus, new strategies for anti-angiogenic cancer therapy based on novel targets are urgently required. Our previous in vitro work suggested that small molecule Cdk5 inhibitors affect angiogenic processes such as endothelial migration and proliferation. Moreover, we recently uncovered a substantial role of Cdk5 in the development of lymphatic vessels. Here we pin down the in vivo impact of endothelial Cdk5 inhibition in angiogenesis and elucidate the underlying mechanism in order to judge the potential of Cdk5 as a novel anti-angiogenic and anti-cancer target. By the use of endothelial-specific Cdk5 knockout mouse models and various endothelial and tumor cell based assays including human tumor xenograft models, we show that endothelial-specific knockdown of Cdk5 results in excessive but non-productive angiogenesis during development but also in tumors, which subsequently leads to inhibition of tumor growth. As Cdk5 inhibition disrupted Notch function by reducing the generation of the active Notch intracellular domain (NICD) and Cdk5 modulates Notch-dependent endothelial cell proliferation and sprouting, we propose that the Dll4/Notch driven angiogenic signaling hub is an important and promising mechanistic target of Cdk5. In fact, Cdk5 inhibition can sensitize tumors to conventional anti-angiogenic treatment as shown in tumor xenograft models. In summary our data set the stage for Cdk5 as a drugable target to inhibit Notch-driven angiogenesis condensing the view that Cdk5 is a promising target for cancer therapy.

Pathways for Oncogenesis in T-Cell Acute Lymphoblastic Leukemia Driven By DNA Methylation and Notch Signaling

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia predominantly found in children that has a worse prognosis than other forms of pediatric leukemia with few options for relapsed disease. These issues lead to a need for new targeted therapies. Many of the genes critical for normal T-cell differentiation are aberrantly expressed when patients develop T-ALL, and this knowledge can serve as the basis for rationale design of targeted therapies. In this list are DNA methyltransferase 3A (DNMT3A), a protein that is responsible for de novo DNA methylation, critical for the function of hematopoietic stem cells (HSCs), and NOTCH1, a protein involved in T-cell signaling and regulating cell fate decisions. Patients that with DNMT3A loss-of-functions mutations driving T-ALL have a worse prognosis than their counterparts with normal DNMT3A function. Previous attempts to directly target the action of NOTCH1 have not led to success, specifically gamma secretase inhibitors, so we aim to define the epigenetic regulation of this pathway and the co-operation between DNMT3A and NOTCH1 mutations may open the door for new therapies in patients with the poorest outcomes.We show here that ablation of Dnmt3a in murine HSCs using Mx1-Cre (Mx1-CRE:Dnmt3afl/fl) results in abnormal T-cell development, leading to an accumulation of progenitors at the double negative 2 (DN2) stage (CD4-, CD8-, c-Kit+, CD44+, CD25+) in a proportion of mice (but not all). In affected mice, although there is a significant accumulation of these progenitors, the downstream population of mature T-cells is essentially unchanged compared to wild-type mice. This developmental arrest is associated with increased expression of the Notch1 signaling pathway. Initially suspected as the mechanism given the native action of Dnmt3a, was differential DNA methylation of Notch1 and its downstream targets such as Hes1 and c-Myc, however, through performed bisulfite sequencing of the promoters for those genes and discovered that there was no difference in DNA methylation between wild-type and Dnmt3a-null thymic progenitor cells. The cellular difference underlying accumulation of this Dnmt3a-null thymic progenitor population appears to be increased resistance to apoptosis, and molecular studies are ongoing to explore the molecular mechanism behind this.The observations that thymic progenitor cells lacking Dnmt3a have modified expression of Notch1, and mutations of both DNMT3A and NOTCH1 have been found in T-ALL patient samples, indicates potential co-operation between these two pathways in T-ALL oncogenesis. To test the biological co-operation between these pathways in vivo, we introduced activated Notch1 intracellular domain (NICD) into wild-type and Dnmt3a-null hematopoietic progenitor cells and assessed the latency to T-ALL by bone marrow transplantation. Introducing NICD into a Dnmt3a-null background significantly accelerated T-ALL oncogenesis (median survival wild-type NICD = 72-days, median survival Dnmt3a-null NICD = 46-days, p < 0.001, Figure 1). To explore the molecular differences leading to enhanced T-ALL pathogenesis in a Dnmt3a-null background, we performed RNA-Seq on T-ALL blasts and found that murine leukemia samples with the combination of Dnmt3a loss-of-function and constitutive Notch1 activation show increased in Jak/Stat pathway signaling compared to T-ALL blasts with native Dnmt3a and normal double positive T-cells (CD8+, CD4+).We hypothesize that Jak/Stat pathway inhibitors could improve outcomes for T-ALL patients with combination DNMT3A loss-of-function and activating NOTCH1 mutations. Ruxolitinib is a well-known JAK1/JAK2 inhibitor used in the treatment of myelofibrosis where it has produced robust results. Given the alterations in Jak/Stat signaling, we plan to induce leukemia by elimination of Dnmt3a using the Cre based model, followed by viral introduction of the Notch1 mutant and then treat mice with ruxolitinib and compare survival. It is our hypothesis that the drug will increase latency to leukemia in this quick to develop murine model. Using developed drugs to target new pathways in T-ALL has the potential to offer new strategies to patients with difficult to treat relapsed disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Notch1 Autoactivation via Transcriptional Regulation of Furin, Which Sustains Notch1 Signaling by Processing Notch1-Activating Proteases ADAM10 and Membrane Type 1 Matrix Metalloproteinase.

Notch1 is an evolutionarily conserved transmembrane receptor involved in melanoma growth. Notch1 is first cleaved by furin in the Golgi apparatus to produce the biologically active heterodimer. Following ligand binding, Notch1 is cleaved at the cell membrane by proteases such as ADAM10 and -17 and membrane type 1 matrix metalloproteinase (MT1-MMP), the latter of which we recently identified as a novel protease involved in Notch1 processing. The final cleavage is γ-secretase dependent and releases the active Notch intracellular domain (NIC). We now demonstrate that Notch1 directly regulates furin expression. Aside from activating Notch1, furin cleaves and activates several proteases, including MT1-MMP, ADAM10, and ADAM17. By chromatin immunoprecipitation and a reporter assay, we demonstrate that Notch1 binds at position -1236 of the furin promoter and drives furin expression. The Notch1-dependent enhancement of furin expression increases the activities of MT1-MMP and ADAM10 but not that of ADAM17, as demonstrated by short hairpin RNA (shRNA) knockdown of furin, and promotes the cleavage of Notch1 itself. These data highlight a novel positive-feedback loop whereby Notch1-dependent furin expression can induce Notch1 signaling by increasing Notch1 processing and by potentiating the activity of the proteases responsible for Notch1 activation. This leads to Notch1 signal amplification, which can promote melanoma tumor growth and progression, as demonstrated by the inhibition of cell migration and invasion upon furin inhibition downstream of Notch1. Disruption of such feedback signaling might represent an avenue for the treatment of melanoma.

Epidermal growth factor receptor status and Notch inhibition in non-small cell lung cancer cells.

BackgroundNotch may behave as an oncogene or a tumor suppressor gene in lung cancer cells. Notch receptor undergoes cleavage by enzymes, including γ-secretase, generating the active Notch intracellular domain (NICD). The aim of the present study was to investigate the effect of DAPT, a γ-secretase inhibitor, in non-small cell lung cancer (NSCLC) cells, as well as the impact of epidermal growth factor (EGF) that is over-expressed by NSCLC cells, on Notch signaling. H23, A549, H661 and HCC827 human NSCLC cell lines were used, expressing various NICD and EGF receptor (EGFR) protein levels.ResultsDAPT decreased the number of H661 cells in a concentration-dependent manner, while it had a small effect on H23 and A549 cells and no effect on HCC827 cells that carry mutated EGFR. Notch inhibition did not affect the stimulatory effect of EGF on cell proliferation, while EGF prevented DAPT-induced NICD decrease in H23 and H661 cells. The type of cell death induced by DAPT seems to depend on the cell type.ConclusionsOur data indicate that inhibition of Notch cleavage may not affect cell number in the presence of EGFR mutations and that EGFR may affect Notch signalling suggesting that a dual inhibition of these pathways might be promising in NSCLC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0196-1) contains supplementary material, which is available to authorized users.

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 &amp;gt;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

Interactions of Melanoma Cells with Distal Keratinocytes Trigger Metastasis via Notch Signaling Inhibition of MITF

The most critical stage in initiation of melanoma metastasis is the radial to vertical growth transition, yet the triggers of this transition remain elusive. We suggest that the microenvironment drives melanoma metastasis independently of mutation acquisition. Here we examined the changes in microenvironment that occur during melanoma radial growth. Weshow that direct contact of melanoma cells with the remote epidermal layer triggers vertical invasion via Notch signaling activation, the latter serving toinhibit MITF function. Briefly, within the native Notch ligand-free microenvironment, MITF, the melanocyte lineage master regulator, binds and represses miR-222/221 promoter in an RBPJK-dependent manner. However, when radial growth brings melanoma cells into contact with distal differentiated keratinocytes that express Notch ligands, the activated Notch intracellular domain impairs MITF binding to miR-222/221 promoter. This de-repression of miR-222/221 expression triggers initiation of invasion. Our findings may direct melanoma prevention opportunities via targeting specific microenvironments.

Cleaved NOTCH1 Expression Pattern in Head and Neck Squamous Cell Carcinoma Is Associated with NOTCH1 Mutation, HPV Status, and High-Risk Features

The Notch pathway is frequently altered in head and neck squamous cell carcinomas (HNSCC); however, the clinical significance of NOTCH1 dysregulation is poorly understood. This study was designed to characterize expression of the transcriptionally active NOTCH1 intracellular domain (NICD1) in HNSCCs and evaluate its association with NOTCH1 mutation status and clinical parameters. IHC for NICD1 was performed on 79 previously sequenced archival HNSCCs with known NOTCH1 mutation status. Three distinct immunohistochemical staining patterns were identified: positive/peripheral (47%), positive/nonperipheral (34%), and negative (19%). NICD1 expression was associated with NOTCH1 mutation status (P < 0.001). Most NOTCH1-wild-type tumors were peripheral (55%), whereas mutated NOTCH1 tumors were most commonly negative (47%). Nonperipheral tumors were more likely than peripheral tumors to have extracapsular spread [adjusted odds ratio (aOR), 16.01; 95% confidence interval (CI), 1.92-133.46; P = 0.010] and poor differentiation (aOR, 5.27; 95% CI, 0.90-30.86; P = 0.066). Negative staining tumors tended to be poorly differentiated (aOR, 24.71; 95% CI, 1.53-399.33; P = 0.024) and were less likely to be human papillomavirus (HPV) positive (aOR, 0.043; 95% CI, 0.001-1.59; P = 0.087). NOTCH1 mutagenesis was significantly associated with HPV status, with NOTCH1-wild-type tumors more likely to be HPV positive than NOTCH1-mutated tumors (aOR, 19.06; 95% CI, 1.31-276.15; P = 0.031). TP53 disruptive mutations were not associated with NICD1 expression or NOTCH1 mutation. In conclusion, NICD1 is expressed in three distinct patterns in HNSCC that are significantly associated with high-risk features. These findings further support a dual role for NOTCH1 as both tumor suppressor and oncogene in HNSCC. Further research is necessary to clarify the role of NOTCH1 in HNSCC and understand the clinical and therapeutic implications therein.

Abstract 19285: Notch1 Haploinsufficiency Increases Risk of Congenital Heart Defects in the Setting of Maternal Diabetes by an Epigenetic Mechanism

Congenital heart disease (CHD) is the most common type of birth defect. Epidemiologic studies have shown the importance of genetic and environmental factors in the multifactorial etiology of CHD. Maternal diabetes mellitus (DM) is one of the non-genetic risk factors that predisposes to CHD predominantly cardiac septation and cardiac outflow tract defects. DM is known to be associated with endothelial cell dysfunction and we recently demonstrated a genetic interaction between endothelial nitric oxide synthase and Notch1, which encodes a receptor that functions in an important cardiac developmental signaling pathway. We hypothesized that maternal DM in the setting of Notch1 heterozygosity of the developing embryo will predispose to CHD. Notch1 +/- embryos (E13.5) exposed to maternal DM demonstrated an increased incidence (86%) of ventricular septal defects compared to wildtype littermates (22%) (Table). Gene expression studies in non-diabetic wildtype, diabetic wildtype and Notch1 +/- embryos showed DM was associated with decreased Notch1 mRNA levels and upregulation in Jarid2, a histone H3K9 demethylase known to regulate Notch1. In H9C2 and endocardial-derived cells and chick embryos, we showed that hyperglycemia led to decreased expression of Notch1 and its downstream targets in a dose-dependent manner. Similarly, Jarid2 mRNA levels increased with high glucose. Furthermore, we found reduced luciferase reporter activity in cells transfected with a constitutively active Notch1 intracellular domain with hyperglycemia. Studies demonstrating the relative enrichment of Jarid2 on Notch1 locus with hyperglycemia by ChIP-qPCR will also be presented. Our findings reveal that maternal DM disrupts cardiac development by deregulating the Notch1 signaling pathway and suggest that this gene-environment interaction is mediated by an epigenetic mechanism involving Jarid2 providing the first mechanistic insights for this association.

Abstract 1945: TORC inhibitors increase the cancer stem cell (CSC) population and Notch signaling in triple negative breast cancer

Abstract Tumor-initiating cells (TICs) or cancer stem cells (CSCs) are resistant to chemotherapy and have been associated with metastatic recurrences and poor patient outcome particularly among patients with triple negative breast cancer (TNBC). Genomic and proteomic data have indicated more than 30% of TNBC patients have PI3K/mTOR pathway lesions making this pathway a promising therapeutic target. Recent publications have demonstrated mechanisms of resistance (JAK2/STAT5 and MYC amplification) to PI3K pathway inhibitors. We hypothesized that resistance to TORC inhibition is due to the survival of a CSC population and that targeting pathways that sustain these cells can provide a significant therapeutic benefit. Treatment of TNBC cell lines with the PI3K/mTOR inhibitor NVP-BEZ235 or the TORC1/2 inhibitor MLN128 resulted in a significant reduction of proliferation in vitro. However, we observed that both BEZ235 and MLN128 enriched for a CSC population as assessed by FACS analysis of cancer stem-like markers and mammosphere formation. This observation was specific to TNBC cell lines since BEZ235 and MLN128 significantly abrogated the CSC population in ER+ (MCF7) and HER2+ (HCC1954) breast cancer cell lines. To determine the mechanisms involved in this CSC enrichment we used a Stem Cell specific PCR Array. We observed an increase in Notch1, FGF1 and ABCG2 mRNA levels in TNBC cells treated with BEZ235 and MLN128. Treatment with these inhibitors also increased the expression of the active Notch intracellular domain, the Notch ligand Jagged1, and the Notch1 target genes Hes1 and Hey1 by qRT-PCR and transcriptional reporter activity. In addition to Hes1 and Hey1, c-myc, another Notch target gene, expression was augmented in 2 of the 3 TNBC cell lines tested. Treament with the γ-secretase inhibitor, DAPT and transfection with Notch1 siRNA oligonucleotides abrogated BEZ235 and INK128-mediated enrichment of CSC populations as measured by FACS analysis and mammosphere formation assays. To determine whether inhibition of either TORC1 or TORC2 enriched for the CSC population, we used RNAi against Rictor (TORC2), Raptor (TORC2) or both. We observed that only the combined knockdown of Rictor and raptor increased the CSC population in TNBC cell lines. These observations suggest that treatment of TNBC harboring PI3K pathway aberrations with TORC1/2 inhibitors results in an initial reduction of tumor burden but do not eradicate the drug-resistant, slow cycling CSC population driven by Notch signaling. Thus, combination of a Notch inhibitor with TORC1/2 inhibitors and chemotherapy may be an effective therapeutic strategy to decrease primary tumor growth and prevent recurrences in patients with TNBC. Citation Format: Neil E. Bhola, Valerie Jansen, Carlos Arteaga. TORC inhibitors increase the cancer stem cell (CSC) population and Notch signaling in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1945. doi:10.1158/1538-7445.AM2014-1945

Abstract 5289: Role of notch3 in epithelial-to-mesenchymal transition of HCC

Abstract Introduction: The epithelial-mesenchymal transition (EMT) program is often activated in cancer cells during invasion and metastasis. During EMT, expression of transcription factors such as Snail and Slug is induced due to the reduction in E-cadherin expression. In addition, matrix metallo-proteinases (MMPs) which digest the various components of extracellular matrix also induced during EMT. Various signaling pathways have been involved in the regulation of EMT program. We hypothesize that notch signaling pathway is a potential regulator of EMT program in HCC. Methods: Lysates from human HCC cell lines, Huh-7, HepG2, and Hep3B, were analyzed for the expression of active notch intracellular domain (NICD) 1-3 proteins by western analysis. To determine the role of NICD3 in cancer invasion and metastasis, siRNA against NICD3 was transfected into HCC cell lines. The effect of depletion of NICD3 in HCC cell lines, epithelial-mesenchymal transition (EMT) markers were measured by western analyses. Results: Varied level of expression of NICD1-3 was observed in HCC cell lines tested. Transfection of siRNA against NICD3 reduced the level of NICD3 but not the NICD1 protein. Western analysis for the EMT markers expression showed an increase in E-cadherin and concomitant reduction in Snail, Slug, Matrix Metallo Proteinases (MMPs) 2 and 9. Conclusions: NICD3 inhibition significantly reduced the expression of markers of metastasis by increasing E-cadherin and suppressing transcription factors Snail, Slug, and MMPs. These observed change in the protein levels are occurred in the absence of NICD3 protein but the presence of NICD1 protein suggesting that NICD3 may play a role in metastasis. Therefore, targeting NICD3 may be potential strategy for treatment of HCC. Citation Format: Selvi Kunnimalaiyaan, T. Clark Gamblin, Muthusamy Kunnimalaiyaan. Role of notch3 in epithelial-to-mesenchymal transition of HCC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5289. doi:10.1158/1538-7445.AM2014-5289