Active Form Of Notch1 Research Articles

SUMO specific peptidase 6 regulates the crosstalk between podocytes and glomerular endothelial cells in diabetic kidney disease

There is increasing evidence that the crosstalk between podocytes and glomerular endothelial cells (GECs) exacerbates the progression of diabetic kidney disease (DKD). Here, we investigated the underlying role of SUMO specific peptidase 6 (SENP6) in this crosstalk. In the diabetic mice, SENP6 was decreased in glomerular tissues and its knockdown further exacerbated glomerular filtration barrier injury. In the mouse podocyte cell line MPC5 cells, SENP6 overexpression reversed HG-induced podocyte loss by suppressing the activation of Notch1 signaling. Notch1 intracellular domain (N1ICD) is the active form of Notch1. SENP6 upregulated the ubiquitination of N1ICD by deSUMOylating Notch1, thereby reducing N1ICD and suppressing Notch1 signaling activation in MPC5 cells. Endothelin-1 (EDN1) is a protein produced by podocytes and has been reported to promote GEC dysfunction. The supernatant from HG-treated MPC5 cells induced mitochondrial dysfunction and surface layer injury in GECs, and the supernatant from SENP6-deficient podocytes further exacerbated the above GEC dysfunction, while this trend was reversed by an EDN1 antagonist. The following mechanism study showed that SENP6 deSUMOylated KDM6A (a histone lysine demethylase) and then decreased the binding potency of KDM6A to EDN1. The latter led to the upregulation of H3K27me2 or H3K27me3 of EDN1 and suppressed its expression in podocytes. Taken together, SENP6 suppressed the HG-induced podocyte loss and ameliorated GEC dysfunction caused by crosstalk between podocytes and GECs, and the protective effect of SENP6 on DKD is attributed to its deSUMOylation activity.

A small molecule inhibitor of Notch1 modulates stemness and suppresses breast cancer cell growth.

Although breast cancer stem cells (BCSCs) are well characterized, molecularly targeting and eradicating this sub-population remains a challenge in the clinic. Recent studies have explored several signaling pathways that govern stem cell activation: We and others established that the Notch1 signaling plays a significant role in the proliferation, survival, and differentiation of BCSCs. Earlier, we reported that a newly developed small molecule, ASR490, binds to the negative regulatory region (NRR: The activation switch of the Notch receptor) of Notch1. In vitro results demonstrated that ASR490 significantly inhibited BCSCs (ALDH+ and CD44+/CD24-) and breast cancer (BC) growth at nM concentrations, and subsequently inhibited the colony- and mammosphere-forming abilities of BCSCs and BCs. ASR490 downregulated the expressions of Notch1 intracellular domain (NICD: The active form of Notch1) and its downstream effectors Hey1 and HES1. Inhibition of Notch1-NICD facilitated autophagy-mediated growth inhibition by triggering the fusion of autophagosome and autolysosome in BCSCs. ASR490 was found to be non-toxic to healthy cells as compared to existing Notch1 inhibitors. Moreover, oral administration of ASR490 abrogated BCSC and BC tumor growth in the in vivo xenograft models. Together our results indicate that ASR490 is a potential therapeutic agent that inhibits BC tumor growth by targeting and abolishing Notch1 signaling in BCSCs and BC cells.

Abstract 3998: Discovery of a small molecule inhibitor for triple-negative breast cancer (TNBC)

Abstract Introduction: Little progress has been made towards developing alternative therapeutic strategies for triple-negative breast cancer (TNBC). Aberrant Notch1 activation, particularly in breast cancer stem cells (BCSCs) in TNBC tumors, enhances this cancer's aggressivity and resistance and consequently contributes to the poor overall survival of patients with TNBC. In contrast, inhibition of Notch1 has been shown to suppress TNBC growth. Thus, Notch1 presents as an attractive target for inhibiting TNBC growth. This study aims to overcome the limitations of current Notch1 inhibitors and identify a more specific inhibitor. Methods: Small molecule screening was performed using structure-activity relationship (SAR) studies, and molecular docking and isothermal calorimetric (ITC) assays were used to determine the target sites of our rationally designed Notch1 inhibitor on the Notch1 receptor. The therapeutic potential of the identified small molecule Notch1 inhibitor was determined on BCSC and TNBC (ALDH+, BCSC, ALDH-, MDA-MB-231) cell lines using molecular studies and in vivo assays. Results: We identified ASR490, a small molecule that binds to the NRR (the activation switch of the Notch receptor) of Notch1. Molecular docking studies indicated a strong interaction between ASR490 and the NRR domain with binding energy of -52.55 kcal/mol. Residue-wise interaction analysis estimated three hydrogen-bond interactions between ASR490 and NRR residues Asn-1483, Glu-1673, and Gly-1664 mediated by water molecules. The finding was further confirmed using biophysical assays (thermal shift assay and isothermal calorimetric analysis) using recombinant Notch1 protein. In vitro results demonstrated that ASR490 significantly inhibited TNBC growth (p <0.001) at nM concentrations, with an IC50 of 760 nM at 24h. Further, the inhibitory effect was confirmed by TNBC’s colony-forming abilities. Western blot analysis confirmed that ASR490 downregulated the expressions of NICD (the active form of Notch1) and its downstream effectors Hey-1 and HES1 by ubiquitinating NRR of Notch1. ASR490 was found to be non-toxic to healthy cells. Moreover, ASR490 exhibited a maximum tolerated dose of more than 100 mg/kg body weight in mice and abrogated TNBC tumor growth in vivo xenograft models. Conclusion: Our results demonstrate ASR490 as a potential therapeutic agent that abolishes Notch1 signaling in TNBC and BCSCs by specifically targeting Notch1 NRR. Further investigation is required for the clinical translational potential of this agent. Citation Format: Uttara Saran, Balaji Chandrasekaran, Ashish Tyagi, Amandeep Singh, Murali K. Ankem, Arun K. Sharma, Chendil Damodaran. Discovery of a small molecule inhibitor for triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3998.

Abstract 3434: A tumor suppressor enhancer of PTEN in T-cell development and transformation

Abstract Recent studies have dissected the mutational landscape of T-cell acute lymphoblastic leukemia (T-ALL), identifying several oncogenes and tumor suppressors implicated in T-cell transformation. However, most genetic lesions in cancer are located in intergenic regions, whose role remains poorly understood. In this context, 20% of T-ALL patients show loss of expression of the tumor suppressor PTEN, but not all can be explained by mutations/deletions of its coding region, defective splicing or promoter hypermethylation. Thus, we hypothesized that alterations of yet unidentified enhancers might play a critical role in the regulation of PTEN expression in T-ALL. To test this, we first performed 4C-seq of the PTEN promoter in human T-ALL cells. Integration of our results with epigenetic profiling revealed a distal non-coding region (named PE for PTEN enhancer) that shows concomitant interaction with the PTEN promoter and bona fide enhancer marks. Moreover, reciprocal 4C-seq assays using PE as viewpoint confirmed its interaction with the PTEN promoter. Next, multispecies DNA sequence alignment revealed notable conservation of PE in mammals and detailed analysis of H3K27ac across 64 cell lines and tissues revealed that PE is specifically active in T-cells and other hematopoietic cells, but is largely repressed in non-hematopoietic cells. Moreover, luciferase assays showed strong, orientation-independent activation of reporter constructs containing either the mouse or human PE sequence in Jurkat cells, and CRISPR/Cas9-induced deletion of PE in human T-ALL cells led to reduced PTEN levels, increased phospho-AKT and enhanced cellular proliferation. To formally test the functional relevance of PE in T-cell development and transformation, we generated PE knockout and conditional knockout mice. PE-null animals are viable and show a marked increase in thymus size and cellularity as their only developmental alteration. Mechanistically, this phenotype is associated with a marked reduction in Pten expression. Next, we analyzed the role of PE in the induction of NOTCH1-driven T-ALLs by transplanting mice with PE wild-type or knockout hematopoietic progenitors infected with retroviruses expressing a mutant constitutively active form of NOTCH1. In this context, mice transplanted with NOTCH-infected PE-null cells showed 100% penetrance and developed T-ALL ~50 days after transplant. However, mice transplanted with NOTCH-infected PE wild-type cells showed delayed T-ALL kinetics and reduced penetrance. In addition, secondary loss of PE in already established NOTCH1-induced T-ALLs from PE conditional knockout mice led to accelerated T-ALL progression and a gene expression signature driven by Pten loss. Finally, analyses of human T-ALL patient samples uncovered recurrent deletions encompassing PE without concomitant deletion of the PTEN coding sequence. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in the pathogenesis of human leukemia. Citation Format: Luca Tottone, Olga Lancho, Maria Victoria da Silva-Diz, Shirley Luo, Shreeta Chakraborty, Pedro P. Rocha, Daniel Herranz. A tumor suppressor enhancer of PTEN in T-cell development and transformation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3434.

Localization of DLL1- and NICD-positive osteoblasts in cortical bone during postnatal growth in rats

The long bone midshaft expands by forming primary osteons at the periosteal surface of cortical bone in humans and rodents. Osteoblastic bone formation in the vascular cavity in the center of primary osteons is delayed during cortical bone development. The mechanisms of the formation of primary osteons is not fully understood, however. Focusing on NOTCH1 signaling, an inhibitory signaling on osteoblastic bone formation, our immunohistochemical analysis revealed Delta like1 (DLL1), a ligand of NOTCH1, and the NOTCH1 intracellular domain (NICD, an activated form of NOTCH1) immunoreactivity, in the cuboidal osteoblasts lining the bone surface in the vascular cavity of primary osteons during postnatal growth in rats. Interestingly, five days after treatment of primary osteoblasts with ascorbic acid and β glycerophosphate, protein levels of both DLL1 and NICD increased transiently, indicating that DLL1 activates NOTCH1 in primary cultured osteoblasts. Thus, the results imply that DLL1-NOTCH1 signaling in osteoblasts is associated with primary osteonal bone formation.

Oncogenic Notch Promotes Long-Range Regulatory Interactions within Hyperconnected 3D Cliques

Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

Notch signaling activation induces cell death in MAPKi-resistant melanoma cells.

The role of Notch signaling in melanoma drug resistance is not well understood. In this study, we show that although NOTCH proteins are upregulated in metastatic melanoma cell lines, Notch signaling inhibition had no effect on cell survival, growth, migration or the sensitivity of BRAFV600E-melanoma cells to MAPK inhibition (MAPKi). We found that NOTCH1 is downregulated in melanoma cell lines with intrinsic and acquired resistance to MAPKi. Forced expression of NICD1, the active form of Notch1, caused apoptosis of the NOTCHlo , MAPKi-resistant cells, but not the NOTCHhi , MAPKi-sensitive melanoma cell lines. Whole transcriptome-sequencing analyses of NICD1-transduced MAPKi-sensitive and MAPKi-resistant cells revealed differential regulation of endothelin 1 (EDN1) by NICD1, that is, downregulation in MAPKi-resistant cells and upregulation in MAPKi-sensitive cells. Knockdown of EDN1 partially mimicked the effect of NICD1 on the survival of MAPKi-resistant cells. We show that the opposite regulation of EDN1 by Notch signaling is mediated by the differential regulation of c-JUN by NICD1. Our data show that MAPKi-resistant melanoma cells acquire vulnerability to Notch signaling activation and suggest that Notch-c-JUN-EDN1 axis is a potential therapeutic target in MAPKi-resistant melanoma.

Maintenance of hematopoietic stem and progenitor cells in fetal intra-aortic hematopoietic clusters by the Sox17-Notch1-Hes1 axis

The aorta-gonad-mesonephros region, from which definitive hematopoiesis first arises in midgestation mouse embryos, has intra-aortic hematopoietic clusters (IAHCs) containing hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We previously reported expression of the transcription factor Sox17 in IAHCs, and overexpression of Sox17 in CD45lowc-KIThigh cells comprising IAHCs maintains the formation of cell clusters and their multipotency in vitro over multiple passages. Here, we demonstrate the importance of NOTCH1 in IAHC formation and maintenance of the HSC/HPC phenotype. We further show that Notch1 expression is positively regulated by SOX17 via direct binding to its gene promoter. SOX17 and NOTCH1 were both found to be expressed in vivo in cells of IAHCs by whole mount immunostaining. We found that cells transduced with the active form of NOTCH1 or its downstream target, Hes1, maintained their multipotent colony-forming capacity in semisolid medium. Moreover, cells stimulated by NOTCH1 ligand, Jagged1, or Delta-like protein 1, had the capacity to form multilineage colonies. Conversely, knockdown of Notch1 and Hes1 led to a reduction of their multipotent colony-forming capacity. These results suggest that the Sox17-Notch1-Hes1 pathway is critical for maintaining the undifferentiated state of IAHCs.

MED12 mutations and NOTCH signalling in chronic lymphocytic leukaemia.

Mutations in the N-terminus of MED12 protein occur at high frequency in uterine leiomyomas and breast fibroepithelial tumours, and are frequently found in chronic lymphocytic leukaemia (CLL). MED12 mutations have been previously linked to aberrant Cyclin C-CDK8 kinase activity, but the exact oncogenic function in CLL is unknown. Here, we characterized MED12 mutations in CLL and identified recurrent mutations in 13 out of 188 CLL patients (6·9%), which clustered in the N-terminus. MED12 mutations were associated with unmutated IGHV (P=0·024). Protein analysis of NOTCH1 in primary CLL samples revealed increased levels of NOTCH1 intracellular domain (NICD), the active form of NOTCH1, in the context of MED12 mutations. We found evidence that NICD is the target of Cyclin C-CDK8 kinase using a specific CDK8 inhibitor. In line with these findings, MED12 mutations were mutually exclusive to mutations in NOTCH1 in CLL, based on a meta-analysis of 1429 CLL patients (P=0·011). Our results suggest that MED12 mutations may contribute to CLL pathogenesis by activating NOTCH signalling.

Abstract 5010: Activation of the notch signaling pathway confers a tumor-suppressive phenotype on melanoma-associated fibroblasts

Abstract Objectives. The tumor microenvironment (TME) is an emerging therapeutic target for cancer treatment. Cancer-associated fibroblasts (CAF) play a crucial role in cancer progression. We aim to target TME by altering intracellular signaling which determines the biological function of CAF. We have recently showed that the Notch signaling pathway likely functions as a molecular switch in controlling the tumor regulatory role of CAF in animal models and experimentally created “CAF”. Here, we investigated the status of Notch signaling in human melanoma-associated fibroblasts (MAF) versus their normal counterparts and tested whether manipulation of the Notch pathway activity in MAF alter their tumor-regulating function. Methods. We examined levels of Hes1, a canonical Notch target, in MAF of human malignant melanoma at different stages (I-IV) and fibroblasts in either adjacent or non-adjacent normal skin tissues using tissue microarray. MAF were isolated from human metastatic melanoma tissues. Notch pathway RT2-PCRArray and immunoblot were used to assess Notch pathway activity in MAF versus normal human dermal fibroblasts. Activation of Notch signaling pathway in MAF was achieved by lentiviral vector encoding active form of Notch1 (NIC). The effect of Notch1-engineered MAF on melanoma growth was tested by in vitro co-cultures and in a mouse co-xenograft model (n=6/group). Tumor angiogenesis was analyzed by immunochemistry. Results. MAF expressed decreased levels of Hes1 compared with adjacent skin fibroblasts. Isolated MAF also exhibited lower Notch activity than normal human dermal fibroblasts. Notch1-engineered MAF significantly inhibited melanoma cell growth in vitro (p<0.01) and suppressed melanoma growth and tumor angiogenesis in mice (p<0.05). Conclusions. Notch pathway activity is down-regulated in MAF. Increase of Notch pathway activity confers MAF with inhibition to melanoma growth and tumor angiogenesis. Our study demonstrates that Notch signaling is a critical molecular switch in determining the tumor regulatory role of MAF and provides potential targets for cancer therapeutic interventions on the TME. Citation Format: Hongwei Shao, Mecker G. Moller, Long Cai, Leiming Zhang, Zhao-Jun Liu. Activation of the notch signaling pathway confers a tumor-suppressive phenotype on melanoma-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5010. doi:10.1158/1538-7445.AM2017-5010

(-)-Epigallocatechin Gallate Targets Notch to Attenuate the Inflammatory Response in the Immediate Early Stage in Human Macrophages.

Inflammation plays important roles at different stages of diabetes mellitus, tumorigenesis, and cardiovascular diseases. (−)-Epigallocatechin gallate (EGCG) can attenuate inflammatory responses effectively. However, the immediate early mechanism of EGCG in inflammation remains unclear. Here, we showed that EGCG attenuated the inflammatory response in the immediate early stage of EGCG treatment by shutting off Notch signaling and that the effect did not involve the 67-kDa laminin receptor, the common receptor for EGCG. EGCG eliminated mature Notch from the cell membrane and the nuclear Notch intercellular domain, the active form of Notch, within 2 min by rapid degradation via the proteasome pathway. Transcription of the Notch target gene was downregulated simultaneously. Knockdown of Notch 1/2 expression by RNA interference impaired the downregulation of the inflammatory response elicited by EGCG. Further study showed that EGCG inhibited lipopolysaccharide-induced inflammation and turned off Notch signaling in human primary macrophages. Taken together, our results show that EGCG targets Notch to regulate the inflammatory response in the immediate early stage.

Endothelial sirtuin 1 inactivation enhances capillary rarefaction and fibrosis following kidney injury through Notch activation

Peritubular capillary (PTC) rarefaction along with tissue fibrosis is a hallmark of chronic kidney disease (CKD). However, molecular mechanisms of PTC loss have been poorly understood. Previous studies have demonstrated that functional loss of endothelial sirtuin 1 (SIRT1) impairs angiogenesis during development and tissue damage. Here, we found that endothelial SIRT1 dysfunction causes activation of endothelial Notch1 signaling, which leads to PTC rarefaction and fibrosis following kidney injury. In mice lacking functional SIRT1 in the endothelium (Sirt1 mutant), kidney injury enhanced apoptosis and senescence of PTC endothelial cells with impaired endothelial proliferation and expanded myofibroblast population and collagen deposition. Compared to wild-type kidneys, Sirt1 mutant kidneys up-regulated expression of Delta-like 4 (DLL4, a potent Notch1 ligand), Hey1 and Hes1 (Notch target genes), and Notch intracellular domain-1 (NICD1, active form of Notch1) in microvascular endothelial cells (MVECs) post-injury. Sirt1 mutant primary kidney MVECs reduced motility and vascular assembly and enhanced senescence compared to wild-type kidney MVECs. This difference in the phenotype was negated with Notch inhibition. Concurrent stimulation of DLL4 and transforming growth factor (TGF)-β1 increased trans-differentiation of primary kidney pericytes into myofibroblast more than TGF-β1 treatment alone. Collectively, these results indicate that endothelial SIRT1 counteracts PTC rarefaction by repression of Notch1 signaling and antagonizes fibrosis via suppression of endothelial DLL4 expression.

MB-01 * NOTCH1 PROMOTES MYC MEDULLOBLASTOMA METASTASIS, INITIATION AND MAINTENANCE

Medulloblastoma (MB) is a malignant brain tumor that originates in the cerebellum in children and spreads via the cerebrospinal fluid to the leptomeningeal spaces of the brain and spinal cord. MB is stratified into four distinct groups, according to genetic and clinical features, and patients from group 3 (also known as c-Myc-amplified) have the highest risk of developing metastatic disease and, consequently, poor prognosis. Treatment protocols involve surgery, craniospinal radiation, and high-dose chemotherapy, which frequently cause disabling neurotoxic effects in long-term survivors. We used MB cell lines and primary cells isolated from patients from the c-Myc-amplified group as tools to understand the cellular determinants of leptomeningeal dissemination. Cells isolated from metastatic tumors express 10 times more NICD1 (Notch1 Intracellular Domain), the active form of Notch1, than cells isolated from primary sites (as quantified by western blot and imunohistochemistry), what implies the upregulation of Notch1 signaling pathway in MB metastasis. Moreover, flow cytometry analyses revealed that the cells isolated from metastatic sites express higher levels of full-length Notch1 when compared to cells from primary sites. These differences occur because of the enrichment in the cancer stem cell population at the metastatic sites, as we observed that MB cells isolated from metastatic tumors exhibit higher self-renewal potential than cells from primary tumor sites.

Endogenous gradients of resting potential instructively pattern embryonic neural tissue via Notch signaling and regulation of proliferation.

Biophysical forces play important roles throughout embryogenesis, but the roles of spatial differences in cellular resting potentials during large-scale brain morphogenesis remain unknown. Here, we implicate endogenous bioelectricity as an instructive factor during brain patterning in Xenopus laevis. Early frog embryos exhibit a characteristic hyperpolarization of cells lining the neural tube; disruption of this spatial gradient of the transmembrane potential (Vmem) diminishes or eliminates the expression of early brain markers, and causes anatomical mispatterning of the brain, including absent or malformed regions. This effect is mediated by voltage-gated calcium signaling and gap-junctional communication. In addition to cell-autonomous effects, we show that hyperpolarization of transmembrane potential (Vmem) in ventral cells outside the brain induces upregulation of neural cell proliferation at long range. Misexpression of the constitutively active form of Notch, a suppressor of neural induction, impairs the normal hyperpolarization pattern and neural patterning; forced hyperpolarization by misexpression of specific ion channels rescues brain defects induced by activated Notch signaling. Strikingly, hyperpolarizing posterior or ventral cells induces the production of ectopic neural tissue considerably outside the neural field. The hyperpolarization signal also synergizes with canonical reprogramming factors (POU and HB4), directing undifferentiated cells toward neural fate in vivo. These data identify a new functional role for bioelectric signaling in brain patterning, reveal interactions between Vmem and key biochemical pathways (Notch and Ca(2+) signaling) as the molecular mechanism by which spatial differences of Vmem regulate organogenesis of the vertebrate brain, and suggest voltage modulation as a tractable strategy for intervention in certain classes of birth defects.

Effects of GLI1 and CTNNB1 Knockdown on Notch Signaling and Proliferation of AML and T-ALL Cell Lines

Objective: Although Hedgehog (Hh) and Wnt signaling pathways are known to play a role in cancer cell growth, their specific effects on leukemia cells are not fully understood. Previously we reported that the Hh inhibitor, cyclopamine, and the Wnt inhibitor, quercetin, suppressed the growth of leukemia cells. However, a specific inhibitory role of these chemical compounds on Hh and Wnt signaling could not be confirmed since these compounds also have off-target effects. Therefore the effects of specific inhibition of Hh and Wnt signaling pathways must be investigated in detail. In our previous study, we reported that small interfering RNA (siRNA)-mediated knockdown of NOTCH1 and NOTCH2 suppressed growth of T-ALL cell lines. In this context, the relationship between Hh, Wnt, Notch and mTOR signaling; known to regulate stemness; has not been fully elucidated. To address these questions, the effects of siRNA-mediated knockdown of GLI1 and CTNNB1 (Catenin beta 1), which respectively mediate Hh and Wnt signaling, on cell proliferation as well as Notch and mTOR signaling were studied in AML and T-ALL cell lines.Methods: Two AML cell lines (NB4 and THP-1) and 2 T-ALL cell lines (DND41 and Jurkat) were used in this study. siRNAs targeting GLI1 (siGL), CTNNB1 (siCT), NOTCH1 (siN1) or control siRNA were transfected into cells by using the pipette tip chamber-based electroporation system. The effects of siGL and siCT transfection on cell proliferation were examined using a colorimetric WST-8 assay and by observing cytospin preparations of the harvested cells. The effects on mRNA and protein expression were examined by quantitative RT-PCR and immunoblotting, respectively.Results: Transfection with siGL and siCT selectively suppressed the expression of GLI1 and CTNNB1, respectively. siRNA treatment did not lead to any significant effects on cell proliferation or morphology in all tested cell lines. In NB4 cells, GLI1 as well as CTNNB1 knockdown increased the level of NOTCH1, the cleaved NOTCH1 fragment (active form of NOTCH1), HES1, and phosphorylated mTOR protein. In the other cell lines, the expression of these proteins was not significantly altered by siGL and siCT.Discussion: siRNA-mediated knockdown experiments suggested that Hh and Wnt signaling had insignificant effect on the proliferation of the leukemia cell lines used in this study. We report a novel interaction, namely, the activation of Notch and mTOR signaling by the suppression of Hh and Wnt signaling in NB4 cells. The molecular mechanism and significance of this phenomenon as well as its translation to other cell lines needs further examination. We propose that this finding must be taken into account when Hh- and Wnt-targeted therapy against leukemia are developed. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 4041: Involvement of Notch1 signaling pathway in medulloblastoma metastasis

Abstract Medulloblastoma (MB) is a malignant brain tumor that originates in the cerebellum in children and spreads via the cerebrospinal fluid to the leptomeningeal spaces of the brain and of the spinal cord. MB is stratified into four distinct groups, according to genetic and clinical features, and patients from group 3 (also known as c-Myc-amplified group) have the highest risk of developing metastatic disease and, consequently, a poor prognosis. Treatment protocols involve surgery, craniospinal radiation, and high-dose chemotherapy, which frequently cause disabling neurotoxic effects in long-term survivors. We used MB cell lines and primary cells isolated from patients from the c-Myc-amplified group to develop a spontaneous spinal metastasis orthotopic xenograft model as a tool to understand the cellular determinants of leptomeningeal and spinal dissemination. Human cells isolated from the primary site tumors expressed 10 fold higher NICD1 (Notch1 Intracellular Domain), the active form of Notch1, than cells isolated from spinal metastatic sites (as quantified by western blot and imunohistochemistry), suggesting the downregulation of canonical Notch1 signaling pathway in MB metastasis. Moreover, flow cytometry analyses revealed that a higher percentage of cells isolated from metastatic sites expressed full-length surface Notch1 as compared to the primary site tumor. These differences cannot be explained by enrichment in the stem cell population at primary tumor sites, as we observed that MB cells isolated from primary tumors and metastatic sites exhibit equivalent self-renewal potential and express equivalent levels of CD133 and CD15. Our goal is to understand the role of notch in regulating medulloblastoma metastasis. Citation Format: Suzana A. Kahn, Sharareh Gholamin, Michael Zhang, Ryan Nitta, Irving Weissman, Siddhartha Mitra, Samuel Cheshier. Involvement of Notch1 signaling pathway in medulloblastoma metastasis. [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 4041. doi:10.1158/1538-7445.AM2014-4041

Activated Notch1 expression is associated with angiogenesis in cutaneous melanoma.

An early event in melanocytic tumor growth is the upregulation of Notch signaling. When an active form of Notch1 is overexpressed in primary human melanocytes, it increases cell growth, survival and invasive properties, promoting melanoma progression. Recent evidence suggested that tumor initiation and growth are driven by a subset of tumor-initiating cells termed cancer stem cells. Notch1 plays a predominant role in the maintenance of melanoblasts, including melanocyte stem cells, by preventing initiation of apoptosis. Moreover, the importance of Notch1 in the regulation of tumor angiogenesis is supported by growing evidence in various cancers. Nestin has been widely used as a marker for melanocyte stem cells as well as an angiogenic marker to evaluate neovascularity of endothelial cells in tumors. To gain an insight into the impact of Notch1 activation on the maintenance of melanocyte stem cells and angiogenesis in melanoma, the expression levels of activated Notch1 and nestin were analyzed by immunohistochemistry in 114 primary cutaneous melanomas and 35 lymph node metastases. Activated Notch1 and nestin expression was also evaluated in four dysplastic melanocytic nevi. This study provides evidence that activated Notch1 is overexpressed in cutaneous melanoma, in tumor cells as well as in microvessel endothelium, and that it can promote tumor angiogenesis. Indeed, the overexpression of activated Notch1 in both tumor and vascular endothelial cells was significantly associated with microvascular density in melanoma samples. Thus, activated Notch1 inhibitors may provide a therapeutic strategy in the treatment of melanoma by blocking tumor-associated vascularization.

Notch1 Activation Up-Regulates Pancreatic and Duodenal Homeobox-1

Transcription factor pancreatic and duodenal homeobox-1 (PDX-1) plays an essential role in pancreatic development, β-cell differentiation, maintenance of normal β-cell function and tumorigenesis. PDX-1 expression is tightly controlled through a variety of mechanisms under different cellular contexts. We report here that overexpression of Notch1 intracellular domain (NICD), an activated form of Notch1, enhanced PDX-1 expression in both PDX-1 stable HEK293 cells and mouse insulinoma β-TC-6 cells, while NICD shRNA inhibited the enhancing effect. NICD-enhanced PDX-1 expression was accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells, which was reversed by NICD shRNA. Cre activation-induced specific expression of NICD in islet β cells of transgenic βNICD+/+ mice induced increased expression of PDX-1, insulin and proliferating cell nuclear antigen (PCNA) and decreased expression of p27 with accompanied fasting hyperinsulinemia and hypoglycemia and altered responses to intraperitoneal glucose tolerance test. Systemically delivered NICD shRNA suppressed islet expression of PDX-1 and reversed the hypoglycemia and hyperinsulinemia. Moreover, expression levels of NICD were correlated with those of PDX-1 in human pancreatic neuroendocrine tumor. Thus, Notch1 acts as a positive regulator for PDX-1 expression, cooperates with PDX-1 in the development of insulin overexpression and islet cell neoplasia and represents a potential therapeutic target for islet neoplasia.

Abstract 3955: The tumor suppressor C/EBPdelta promotes hypoxia-induced EMT and expression of stem cell markers through inhibition of FBXW7: Implications for mammary tumor metastasis.

Abstract The transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) exhibits tumor-suppressor like-qualities such as downregulation of cyclin D1, and promotion of growth arrest and apoptosis. However, using a mouse model of mammary tumorigenesis (MMTV-Neu), we had found that C/EBPδ has dual functions. C/EBPδ suppresses primary tumor incidence while also augmenting metastatic progression. The pro-metastatic function of C/EBPδ can be correlated with a role in hypoxia adaptation and expression of pro-inflammatory genes and macrophage activation. C/EBPδ exerts both of these functions through inhibition of FBXW7, a component of SCF E3 ubiquitin ligase complex, which targets substrates such as c-myc and Notch for degradation (Balamurugan et al, 2010; Balamurugan et al., under revision). Hypoxia is believed to promote tumor progression in part by inducing epithelial-mesenchymal transition (EMT), which contributes to the generation of stem-like cancer cells (CSCs). In the present study, we found that hypoxia-induced EMT and expression of CSCs markers were compromised in Cebpd null primary tumor cells and in human breast tumor cells when C/EBPδ expression was silenced. On the other hand, silencing of the C/EBPδ-repressed gene FBXW7 was sufficient to induce a mesenchymal-like phenotype. Loss of FBXW7 has been shown to promote acquisition of stemness. In line with this notion, we found that overexpression of FBXW7 in the breast tumor cell line MDA-MB-468 reduced expression of aldehyde dehydrogenase 1 (Aldh1), C/EBPδ and the active form of NOTCH (NICD). Together, these data suggest that C/EBPδ augments - whereas its target FBXW7 prevents - EMT and breast cancer stem cell development, which may be an additional mechanism by which C/EBPδ promotes metastatic progression. Our current investigations are aimed at understanding how the different functions of C/EBPδ are integrated depending on cellular environment and context that results in its tumor promoting function despite its tumor suppressor role. Citation Format: Balamurugan Kuppusamy, Esta Sterneck. The tumor suppressor C/EBPdelta promotes hypoxia-induced EMT and expression of stem cell markers through inhibition of FBXW7: Implications for mammary tumor metastasis. [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 3955. doi:10.1158/1538-7445.AM2013-3955

Abstract 1636: Altering the function of cancer-associated fibroblasts via activation of Notch signaling to inhibit tumor growth.

Abstract Objectives. Cancer-associated fibroblasts (CAF) play critical roles in determining tumor growth, invasion, metastasis, drug response and tumor angiogenesis. We have previously demonstrated that activation of the Notch1 pathway confers normal human dermal fibroblasts a suppressive phenotype to melanoma growth. Here, we further investigated whether manipulation of Notch signaling in melanoma-associated fibroblasts (MAF) alter their regulatory phenotype by which affects melanoma growth. Methods. MAF were isolated from human metastatic melanoma and characterized. Notch pathway activity in MAF versus normal human dermal fibroblasts was examined using Notch pathway RT2-PCRArray and immunoblotting. Enforced activation of Notch pathway was achieved by transducing MAF with lentiviral vector encoding active form of Notch1 (NIC). The effect of active Notch1-engineered MAF on melanoma growth was tested by in vitro co-culture and in vivo co-xenografting animal model. Results. Isolated MAF are α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) positive. MAF exhibited a relatively lower Notch activity compared to normal human dermal fibroblasts. Enforced activation of Notch1 pathway suppressed cellular activities of MAF. Active Notch1-engineered MAF significantly inhibited melanoma cell growth in vitro and retarded xenografted human melanoma cell growth on mouse skin. Conclusions. Notch pathway activity is lower in MAF. Constitutive activation of Notch pathway confers MAF to a suppressive phenotype in regulating melanoma growth. Our study demonstrated that Notch signaling functions as a critical molecular switch in determining the regulatory role of tumor stromal fibroblasts and provided a novel approach to target tumor microenvironment through modulating stromal fibroblasts by manipulation of Notch signaling. Citation Format: Hongwei Shao, Mecker G. Moller, Omaida C. Velazquez, Zhao-Jun Liu. Altering the function of cancer-associated fibroblasts via activation of Notch signaling to inhibit tumor growth. [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 1636. doi:10.1158/1538-7445.AM2013-1636