Notch1 mRNA Research Articles

Biological therapy induces expression changes in Notch pathway in psoriasis.

Psoriasis is a chronic inflammatory skin disease, characterized by hyperproliferation of keratinocytes and by skin infiltration of activated T cells. To date, the pathophysiology of psoriasis has not yet been fully elucidated. The Notch pathway plays a determinant role in cell fate determination, proliferation, differentiation, immune cell development and function. Many biological agents, used in the treatment of psoriasis, include TFN-α inhibitors, such as etanercept, adalimumab, and anti IL-12/IL-23 p40 antibody, such as ustekinumab. This study aimed to determine mRNA expression levels by real-time RT-PCR, and protein expression levels, analysed by Western blot and immunohistochemistry, of some components of the Notch pathway, such as NOTCH1, NOTCH2, JAGGED1, and HES1 after biological treatments in psoriatic patients. mRNA and protein levels of NOTCH1, NOTCH2, JAGGED1 and HES1 were upregulated in skin samples from untreated psoriatic patients compared with normal controls. Biological therapy showed to downregulate differently the protein expression levels of the molecules under study. Our study suggests that Notch pathway components might be a potential therapeutic target against psoriasis.

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

Abstract 2130: The RNA-binding protein tristetraprolin controls intestinal cell differentiation and tumorigenesis through the Notch signaling pathway

Abstract Colorectal cancer (CRC) is the 2nd leading cause of cancer death in U.S. With lifetime incidence rates as high as 1 in 20, it is critical to better define the molecular events controlling intestinal homeostasis and tumor initiation/progression. In normal intestinal homeostasis, the Notch pathway plays a fundamental role in controlling epithelial cell fate through juxtacrine signaling between membrane-bound ligands to the Notch receptor present on contacting cells. This complex promotes intestinal epithelial cell fate through transcription factors Hes1 and Atoh1/Math1, allowing for differentiation of absorptive enterocyte lineage by Hes1 or secretory (goblet, enteroendocrine and Paneth) cell lineage controlled by Atoh1. In CRC however, constitutive Notch activation is observed allowing for enhanced Hes1 expression associated with tumor progression and self-renewal of tumor-initiating cells. Yet, a clear mechanism for the constitutive activation of this pathway in CRC is not known. Here, we investigated the regulation of Notch signaling by the mRNA decay factor Tristetraprolin (TTP) in CRC and TTP knockout mice. TTP is a member of the TIS11 family of RNA-binding proteins, and the major factor promoting decay of mRNAs bearing AU-rich element (ARE) motifs in their 3′UTR. TTP plays an essential role in controlling pro-inflammatory and oncogenic gene expression in the GI tract, however during CRC progression, TTP expression is silenced in tumors. In this study, TTP-inducible CRC cells were used to show that expression of TTP suppresses proliferation and anchorage-independent growth, impacts cell cycle, and reduces xenograft tumor growth. Interestingly, TTP expression altered CRC cells such that cells now displayed a secretory cell-like morphology, consistent with studies showing effects of Notch signaling attenuation. Alcian blue staining of acidic mucins was significantly increased in TTP-expressing CRC cells and in xenograft tumors, suggesting a role for TTP in directing secretory cell differentiation. Furthermore, gene expression arrays and reporter gene assays showed that levels of Notch signaling genes were significantly impacted by TTP and TTP preferentially promoted a 3-fold downregulation of Notch1 receptor as compared to Notch2-4. The ability of TTP to target Notch1 mRNA for rapid decay occurred through its ARE-containing 3′UTR as observed using 3′UTR reporter and RNA-binding assays. To determine if these effects were specific to tumor cells, intestinal cell lineage was examined in TTP knockout mice with a remarkable reduction in goblet cell number observed as compared to wild-type mice. These findings demonstrate a novel role for TTP in controlling intestinal cell differentiation via post-transcriptional regulation of Notch1 and provide mechanistic insights into constitutive Notch activation observed in CRC occurring through loss of TTP expression. Citation Format: Shufei Zhuang, Liangyan Hu, Sandhya Sanduja, Shahid Umar, Shrikant Anant, Dan A. Dixon. The RNA-binding protein tristetraprolin controls intestinal cell differentiation and tumorigenesis through the Notch signaling pathway. [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 2130. doi:10.1158/1538-7445.AM2015-2130

Emodin inhibits the proliferation of PC3 prostate cancer cells in vitro via the Notch signaling pathway.

The aim of the present study was to examine the anticarcinogenic effects of emodin on the Notch signaling pathway and vascular endothelial growth factor (VEGF) in the PC3 androgen-independent prostate cancer cell line in vitro. The cell viability was assessed using an 3‑(4,5)‑dimethylthiahiazo(‑z‑y1)‑3,5‑di‑phenytetrazolium bromide assay. Cell apoptosis and cell cycle were detected using flow cytometry. Morphological alterations were observed using transmission electron microscopy. The mRNA and protein expression levels of Notch1, Jagged1, VEGF and bFGF were detected using reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. Laser scanning confocal microscope (LSCM) immunofluorescence analysis was performed to detect the levels of expression and the sub‑cellular localization of the Notch1 receptor protein. Growth of the PC3 cells was inhibited by emodin. Flow cytometry demonstrated that emodin induced apoptosis in the PC3 cells and arrested the cell cycle of the PC3 cells at the G2/M phase. The mRNA and protein expression of Notch1 in the PC3 cells was markedly increased, whereas the mRNA and protein expression levels of Jagged1, VEGF and bFGF were significantly decreased following exposure to emodin for 24 h. The LSCM assay revealed that the Notch1 was not only localized in the membrane and cytoplasm, but was also present in the nucleolus of the PC3 cells, and the expression of Notch1 in the nuclei gradually increased following treatment with emodin. These results demonstrated that emodin suppressed the growth of androgen‑independent prostate cancer cell lines and induced apoptosis and cell cycle arrest. The Notch signaling pathway was activated in the PC3 cells following exposure to emodin, which suggested that the Notch signaling pathway is involved in the prostate tumor‑suppressing mechanisms of emodin, the activation of which may depend on CBF1 protein in the nucleus by classic pathways. The antitumor function of emodin, attributed to the Notch signaling pathway, induced the downregulation of VEGF by suppressing tumorigenesis and angiogenesis, which indicated a novel mechanism underlying the emodin-mediated anti-prostate cancer effect.

Construction and identification of competitive 0610009E02Rik long noncoding RNA recombinant adenovirus shuttle vector

Objective To construct a competitive adenovirus vector against 0610009E02Rik, a long noncoding RNA (lncRNA) for investigating its role in the repairment of injured hepatocytes during hepatic veno-occlusive disease (HVOD). Methods Firstly, genome DNA was extracted from the tail of C57BL/6 mice, and amplified the overlapping region of the gene 0610009E02Rik and the 34th exon of Notch1 by polymerase chain reaction(RT-PCR), then it was linked into the adenovirus shuttle plasmid GV359 which was digested by BamHⅠ/NheⅠrestriction enzyme. Secondly, the constructed shuttle plasmid and packaging plasmid pBHG were co-transfected into HEK293 cells for assembly of recombination adenovirus, and subsequent amplification, followed by cesium chloride (CsCl) gradient ultracentrifugation for purification and measurement of the viral titer in HEK 293 cells. Finally, the recombination adenovirus was transfected into hepatocyte cell line H2.35 with different multiplicity of infection (MOI), and transfection efficiency was evaluated by fluorescence microscope. Meanwhile, real-time polymerase chain reaction (RT-PCR) was used to detect the expression of anti-lncRNA, Notch1 and 0610009E02Rik. Western blotting was used for the detection of the expression level of Notch1. Results ① It was discovered that the gene 0610009E02Rik and the 34th exon of Notch1 have the overlapping region (its fragment length is about 1 000 bp), and then it was obtained by PCR with the primer which has the BamHⅠ/NheⅠdigestion sites, and DNA sequencing result confirmed that the competitive adenovirus shuttle plasmid against 0610009E02Rik was successfully constructed. ② At the 12th day after transfection, about 90% cells presented cytopathic effect (CPE), then all the transfected cells were obtained a large amount of virus extract. Then HEK293 cells were transfected by the condensed and purified virus with a series of dilution, and cells transfected with dilution fold of 1∶(10~1010) were all had CPE, and this is verifi the virus titers was 5.01x109 PFU/mL. ③ Different MOI with different transfection efficiency in hepatocytes was observed and transfection efficiency of hepatocytes was about 95% after 48 h with MOI of 80. Real-time PCR was adopted to detect the relative RNA expression levels. anti-0610009E02Rik lncRNA level were (3.13±0.83) folds higher than that of the control groups (P=0.047). Notch1 mRNA relative expression level were (0.38±0.08) folds higher than that of the control group (P=0.010), and 0610009E02Rik lncRNA expression level were (1.04±0.26) folds higher than that of the control groups without significantly differences (P>0.05). Moreover, the protein level of Notch1 was lower than that of control group, and this is consistent with its mRNA level. Conclusion We successfully constructed the competitive adenovirus vector against 0610009E02Rik, which could down-regulate Notch1 mRMA and protein expression in hepatocyte cell line H2.35, providing a basis for further investigation of its role in the repairment of injury hepatocytes during HVOD. Key words: RNA, long noncoding; Receptor, Notch1; Adenovirus, human; DNA, recombinant; hepatocytes

Downregulation of notch signaling pathway in late preterm and term placentas from pregnancies complicated by preeclampsia.

Preeclampsia (PE) is a major cause of maternal mortality and morbidity, affecting 3–5% of all pregnancies. The Notch signaling pathway plays an important role during placental development, activating several target genes. Defects in the Notch pathway have adverse effect on placentation. The aim of this study was to investigate the expression of receptors NOTCH1,-2,-3,-4, ligands DLL1,-3,-4, JAG1,-2 and target genes HEY1,-2 in placental tissue samples from 20 late preterm or term pregnancies complicated by PE versus 20 normal pregnancies. mRNA levels of the studied molecules were measured by quantitative Real-Time PCR (qRT-PCR), while the protein expression of the intracellular domain of NOTCH2 (NICD2) and NOTCH3 (NICD3) was measured by Western Blot (WB). qRT-PCR analysis revealed that NOTCH1, NOTCH4 and DLL1 were not expressed in the placenta. On the contrary, NOTCH2, NOTCH3, DLL3, DLL4, JAG1, JAG2, HEY1 and HEY2 mRNA levels were downregulated in PE samples vs. controls (p<0.01). WB confirmed that NICD2 (p = 0.014) and NICD3 (p<0.001) protein levels were also lower in PE specimens. Statistical analysis revealed several significant associations: of NOTCH3 mRNA expression with smoking during pregnancy (p = 0.029), of NICD3 protein levels (p = 0.028) and DLL3 mRNA levels (p = 0.041) with birth weight centile, and of HEY2 transcript levels with parity (p = 0.034) and mode of delivery (p = 0.028). Our results suggest that Notch pathway downregulation is associated with PE. Further studies are required in order to determine the role of these molecules in PE pathogenesis and to evaluate their potential use for the early detection and treatment of PE.

Interference of Notch1 inhibits the growth of glioma cancer cells by inducing cell autophagy and down-regulation of Notch1-Hes-1 signaling pathway.

Glioma is the most common malignant tumors in adult brains, and Notch signaling pathway plays an important role in cell differentiation. The aim of the present study is to investigate the role of Notch1 in the progression of glioma cancers and clarify the mechanism of Notch1 silencing on inhibiting the proliferation of glioma cancer cells. First, endogenous Notch1 expression was interfered with a lentiviral vector of Notch1 shRNA. RT-PCR and western blotting were used for detecting the expression of Notch1 mRNA and protein, respectively. MTT assay results demonstrated that transfection with Notch1 shRNA and treatment with MRK003, a Notch1 inhibitor, both inhibited the proliferation of glioma cancer cells (p < 0.01). The lentiviral vector of Notch1 shRNA transfected into U251 cells induced cell cycle arrest at G0/G1 phase by FACS with PI staining analysis. Meanwhile, the expression levels of LC3-II and Beclin1 significantly increase in Notch1 shRNA-transfected U251 cells, suggesting that cell autophagy was induced when interfering with Notch1 in glioma cells. The downstream transcription factors were also detected by RT-PCR and western blotting analysis, and the data showed that interference with Notch1 increased the expression level of Hes-1, but not Hes-5. Taken together, all the data obviously revealed that Notch1 played an important role in the progression of glioma cancers. The clarification of the mechanism will be helpful for the diagnosis of glioma cancer and would provide new clues to molecular targets for cancer therapy.

Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast

IntroductionNotch signalling has been shown to control cytotrophoblast (CTB) proliferation, differentiation and motility suggesting that the conserved signalling pathway could be critical for human placental development. Since individual Notch receptors have not been elucidated, we herein investigated expression pattern and function of Notch2 in different first trimester trophoblast subpopulations. MethodsLocalisation of Notch2 was analysed in first trimester placental and decidual tissues using immunofluorescence. Notch2 transcript and protein levels were studied by qRT-PCR and Western blotting in proliferative EGF receptor (EGFR)+ and differentiated HLA-G+ CTBs, respectively, isolated from early placentae by MACS. CTB migration through fibronectin-coated transwells as well as proliferation (EdU labelling) in floating villous explant cultures and primary CTBs were investigated in the presence of Notch2 siRNAs or specific antibodies blocking Notch2 cleavage. ResultsIn tissue sections Notch2 expression was higher in HLA-G+ distal cell column trophoblasts (dCCTs) compared to proximal CCTs. Accordingly, expression of Notch2 mRNA and protein were elevated in isolated HLA-G+ CTBs compared to EGFR+ CTBs. Notch2 was also detectable in interstitial CTBs as well as in intramural CTBs associated with maternal decidual vessels. Antibody-mediated inhibition of Notch2 signalling did not affect proliferation, but increased migration of SGHPL-5 cells and primary CTBs. Similarly, Notch2 siRNA treatment promoted trophoblast motility. DiscussionNotch2 is present in differentiated cells of the extravillous trophoblast lineage, such as dCCTs, interstitial and intramural CTBs, suggesting diverse roles of the particular receptor. Notch2 signalling, activated by cell–cell contact of neighbouring dCCTs, could attenuate trophoblast migration.

Notch ligand Delta-like 1 promotes in vivo vasculogenesis in human cord blood–derived endothelial colony forming cells

Background aimsHuman cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Because Notch signaling is critical for embryonic blood vessel formation in utero, we hypothesized that Notch pathway activation may enhance cultured ECFC vasculogenic properties in vivo. MethodsIn vitro ECFC stimulation with an immobilized chimeric Notch ligand (Delta-like1ext-IgG) led to significant increases in the mRNA and protein levels of Notch regulated Hey2 and EphrinB2 that were blocked by treatment with γ-secretase inhibitor addition. However, Notch stimulated preconditioning in vitro failed to enhance ECFC vasculogenesis in vivo. In contrast, in vivo co-implantation of ECFCs with OP9-Delta-like 1 stromal cells that constitutively expressed the Notch ligand delta-like 1 resulted in enhanced Notch activated ECFC-derived increased vessel density and enlarged vessel area in vivo, an effect not induced by OP9 control stromal implantation. ResultsThis Notch activation was associated with diminished apoptosis in the exposed ECFC. ConclusionsWe conclude that Notch pathway activation in ECFC in vivo via co-implanted stromal cells expressing delta-like 1 promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC.

Electroacupuncture Suppressed Neuronal Apoptosis and Improved Cognitive Impairment in the AD Model Rats Possibly via Downregulation of Notch Signaling Pathway.

Acupuncture is a potential strategy for the treatment of Alzheimer's disease (AD) and the possible mechanisms worth to be explored. In this study, we proposed and tested the hypothesis that whether Notch signaling pathway is involved in the effect of electroacupuncture (EA) treatment. Rats that received EA treatment on the acupoints of Baihui (Du 20) and Shenshu (BL 23) had shorter latency and remained in the original platform quadrant longer and crossed the former platform contained quadrant more frequently compared to the Aβ injection rats without EA treatment. EA obviously alleviated the cell apoptosis resulted by Aβ infusion in hippocampus CA1 regions through upregulating the expression of Bcl-2 and downregulating the expression of Bax. EA could further obviously promote the expression of synapsin-1 and synaptophysin in hippocampus. Aβ injection significantly increased the expression of Notch1, Jag1, and Hes1 mRNA, while EA treatment downregulated the level of Notch1 and Hes1 mRNA in hippocampus, but not Jag1 mRNA. Our data suggested that EA treatment improved learning and memory function in the AD rat model partially through downregulating Notch signaling pathway.

Expression of NOTCH4 affects vasculogenic mimicry formation in hepatocellular carcinoma

Objective To investigate the effect of NOTCH4 expression on vasculogenic mimicry( VM) formation in hepatocellular carcinoma( HCC). Methods Tumor tissues were collected from 85 patients diagnosed with HCC. The relationship between NOTCH4 expression and VM was examined using immunohistochemical staining and PAS / CD31 double staining. The influence of siRNA- mediated NOTCH4 silencing on VM network formation in HCC was observed in the 3D cell culture system in vitro. Experimental data were assessed with Mann-Whitney U test and one- way analysis of variance. Results NOTCH4 had significantly higher expression in VM- positive HCC tissues than in VM- negative HCC tissues( P = 0. 019). In siRNA- treated cells,NOTCH4 mRNA and protein expression was significantly down- regulated compared with those in mock- treated cells( P = 0. 007; P = 0. 003) and untreated cells( P = 0. 000; P = 0. 000). VM network formation was impaired by inhibition of NOTCH4 expression in the 3D cell culture system. Conclusion NOTCH4 plays an important role in VM formation in HCC and may provide a novel molecular target for anti- angiogenesis therapy for HCC.

Hybrid Microrna Control of Colon Cancer Stem Cell Asymmetric Division

Colon cancer stem cells (CCSC) undergo both symmetric and asymmetric division, which balance differentiation versus self-renewal in the tumor cell population. This decision is determined by the microRNA miR-34a, whose spatial segregation generates a bimodal Notch response that determines cell fate outcomes. This bimodal response is caused by kinetic mutual sequestration between miR-34a and Notch mRNA. However, three questions have remained since we reported the above findings (Bu et al, Cell Stem Cell, 2013). First, does miR-34a generate bimodal responses from all target genes? Second, what is the relationship between miR-34a and the canonical cell fate determinant protein Numb, which also targets Notch to regulate cell fate symmetry? And third, do these mechanisms exist in normal stem cells? (1) A systematic study demonstrates that the kinetics of microRNA regulation is target-specific. Quantitative single-cell analysis revealed that miR-34a generates bimodal responses from a small subset of genes that are involved in cell fate determination, but regulating the majority of genes (e.g., metabolic and growth genes) in a non-bimodal manner. (2) We report that miR-34a and Numb directly interact to form an incoherent feedforward loop. This microRNA-protein hybrid circuitry synergistically enhances Notch and cell fate asymmetry by orders of magnitude and exhibits adaptive behavior to offset interference from other miR-34a target genes, hence buffering asymmetric cell fate outcomes from fluctuations in miR-34a levels. (3) We show that the hybrid control mechanism is active in the intestinal stem cell niche. Using innovative abdominal window and 3-D-printed intestinal insert, chronic in vivo multiphoton imaging further revealed the real-time spatiotemporal dynamics of the hybrid circuitry in live mice. This control is gradually subverted in late-stage cancer stem cells, contributing to their proliferation and malignancy.

Compressive Stress Enhances NOTCH1 mRNA Expression in Human Deciduous Dental Pulp Cells in vitro

Compressive Stress Enhances NOTCH1 mRNA Expression in Human Deciduous Dental Pulp Cells in vitro

Haploinsufficiency of the c-myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53-dependent T-cell acute lymphoblastic leukemia progression by activating Notch1.

FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR⁺/⁻) C57BL6 mice were generated. FIR complete knockout (FIR⁻/⁻) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR⁺/⁻ mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR⁺/⁻TP53⁻/⁻ generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR⁺/⁻TP53⁻/⁻ compared with that in FIR⁺/⁺TP53⁻/⁻ mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion, the alternative splicing of FIR, which generates FIRΔexon2, may contribute to both colorectal carcinogenesis and leukemogenesis.

Hematopoietic Stem and Progenitor Cells in Patients with Mature B-Cell Malignancies Are Quantitatively and Qualitatively Deregulated Compared to Healthy Controls

▪Mature B-cell lymphomas represent a heteregenous group of malignancies, which are considered to arise along different steps of B-cell development, particularly from germinal or post-germinal center B-cells. Recent studies, however, indicate that hematopoietic stem and progenitor cells (HSPC) could be involved in the pathogenesis of these diseases.We analyzed HSPC populations from 131 patients with mature B-cell malignancies, including chronic lymphocytic leukemia (CLL, n=20), mantle cell lymphoma (MCL, n=26), diffuse large B-cell lymphoma (DLBCL, n=35), follicular lymphoma (FL, n=25), and multiple myeloma (MM, n=25). For comparison, HSPC populations obtained from 22 healthy donors were used. Hematopoietic stem cells (HSC, Lin-CD34+CD38-CD90+CD45RA-), multipotent progenitors (MPP, Lin-CD34+CD38-CD90-CD45RA-), multi-lymphoid progenitors (MLP, Lin-CD34+CD38-CD90-CD45RA+), and pro-B cells (CD34+CD38+CD10+CD19+) were analyzed by flow cytometry. Proportions of HSPC and pro-B cells were related to CD34+CD38- and CD34+CD38+ cells, respectively. HSC and pro-B cell populations were sorted directly into tubes containing lysis solution for subsequent gene expression analyses. Preamplified cDNA was used for quantitative PCR of selected gene targets (n=28 in HSC, and n=27 in pro-B). The expression of targets was normalized to GAPDH expression. Corresponding cell populations isolated from healthy donors were used as controls.The aforementioned flow cytometry analyses revealed significantly decreased MLP population in all 5 tested diagnoses: CLL (p<0.0001), MCL (p=0.0004), DLBCL (p=0.002), FL (p=0.004), and MM (p=0.004) compared to CTRL (Figure 1). Pro-B cell populations were also decreased in all tested diagnoses, but statistical significance was reached only in MCL (p<0.0001), DLBCL (p=0.0003), and MM (p=0.0025), but not in CLL (p=0.078) or FL (p=0.308). HSC populations were significantly increased in DLBCL (p=0.0023), FL (p=0.015), and MCL (p=0.036), but not in MM (p=0.207) or CLL (p=0.875). The only significant change in MPP population was an increase in CLL (p=0.031).To gain more insight into biology of the stem and progenitor cell populations we analyzed gene expression changes of the key transcriptional regulators, HSC and pro-B cell specific surface markers, or genes that are overexpressed in particular lymphoma subtypes. We observed: 1) Significant upregulation of BCL11A, RUNX1, IKAROS, GATA2, PROM1, and CD44 mRNA within HSC populations obtained from all tested diagnoses compared to CTRL-HSC. 2) None of the targets in pro-B cells were significantly deregulated across all 5 diagnoses, the only significantly upregulated genes were IKAROS and EBF1 in CLL compared to CTRL pro-B cells. 3) NOTCH1 and CCND1 mRNA were not detected in CTRL-HSC, but NOTCH1 was detectable in more than 50% of DLBCL and MM-HSC samples, while CCND1 was detectable in more than 50% of CLL-HSC samples. 4) More than 50% difference in gene expression frequency between CTRL and patient HSC samples was observed in the case of BMI1 (CLL, DLBCL, FL and MM), FOXO1 (MM), FOXP1 (DLBCL), and IRF4 (CLL). 5) Pro-B cells obtained from CTRL samples did not express BCL2, BMI1, MYC, PAX5, or ZAP70, but these genes were detected in more than 50% of patient pro-B cell samples as follows: BCL2 in FL, BMI1 and ZAP70 in CLL and DLBCL, MYC in CLL, DLBCL and FL, PAX5 in CLL. 6) More than 50% difference in gene expression frequency between CTRL and patient pro-B cells was observed in case of BCL11A (CLL, DLBCL, FL, MM, and MCL), BCL2L1 (CLL, DLBCL, FL, MM), CD38 (CLL, DLBCL, FL), CD44 (in CLL, DLBCL), IRF4 (CLL, DLBCL, FL), IRF8 and LEF1 (CLL), PU.1 (CLL, FL), and RUNX1 (CLL, DLBCL).In this study, we showed that bone marrow HSPC in patients with mature B-cell malignancies are quantitatively and qualitatively different compared to HSPC obtained from healthy controls. We currently study whether this deregulation is caused by the influence of malignant cells on HSPC or reflects HSPC intrinsic changes that are involved in the pathogenesis of these malignancies.Grant support: IGA-MZ NT13201-4/2012, GACR14-19590S, UNCE 204021, SVV-2013-266509, GA-UK 595912, PRVOUK-27/LF1/1 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effect of silencing NOTCH1 gene by shRNA interference on AKT/mTOR pathway in mantle cell lymphoma

The study was purposed to investigate the effect of silencing NOTCH1 gene by shRNA interference on the proliferation, apoptosis and the expression of AKT signaling pathway-related proteins in mantle cell lymphoma Jeko-1 cell line. The hairpin-like oligonucleotide sequences targeting NOTCH1 gene were designed and transfected into Jeko-1 cells by lipofectamine (TM) 2 000. NOTCH1 mRNA and protein were detected respectively by RT-PCR and Western blot. Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expressions of BCL-2, BAX, procaspase-3, procaspase-9, Akt, p-Akt, p-mTOR, p-P70S6K were detected by Western blot. The results showed that NOTCH1 mRNA expression was markedly suppressed by the shRNA targeting NOTCH1. NOTCH1 shRNA suppressed the proliferation of Jeko-1 cells and induced apoptosis of these cells. The cell apoptotic rate was (34.5 ± 3.4)%, (2.4 ± 1.3) %, (1.7 ± 0.6) % in NOTCH1 shRNA, Neg-shRNA and blank groups, respectively, and the difference between them was statistically significant (P < 0.01). NOTCH1 shRNA down-regulated the expression of BCL-2, procaspase-3, procaspase 9, p-Akt, p-mTOR and p-70S6K, up-regulated the expression of BAX, but no change protein expression of Akt was observed. It is concluded that the silencing NOTCH1 gene expression by shRNA interference may inhibit Jeko-1 proliferation, induce the cell apoptosis, and the mechanisms may be associated with the inhibition of Akt/mTOR signaling pathway by dephosphorylation.

Abstract 18010: Induced Pluripotent Stem Cell-Based Modeling of Human NOTCH1 Mutations Reveals Novel Pathways Regulating Aortic Valve Disease

In humans, NOTCH1 mutations result in congenital heart defects including valve malformations and severe valve calcification in adults. Valve calcification typically occurs on the aortic side of the valve leaflets that is not exposed to laminar shear stress, suggesting a protective role of flow sensed by the endothelial lining. To understand the mechanisms by which NOTCH1 mutations in endothelial cells (ECs) cause disease, we generated induced pluripotent stem cell (iPSC) lines from fibroblasts of four individuals from two families affected with aortic valve disease due to heterozygous non-sense mutations in NOTCH1. We differentiated control and mutant iPSC lines into ECs and exposed the ECs to either static or fluid shear stress conditions. NOTCH1 mRNA levels were decreased by ~50% in the NOTCH1+/- ECs under static or shear stress conditions. RNA-seq revealed that 165 genes were differentially expressed in static conditions and 193 genes responded abnormally to shear stress in NOTCH1+/- ECs compared to NOTCH1+/+ ECs. Differentially expressed genes included canonical NOTCH1 targets HRT2 and EFNB2 as well as novel targets involved in vascular development, inflammation, and endochondral ossification. Anti-calcific genes uniquely upregulated in shear stress were dysregulated in NOTCH1+/- ECs, indicating that they were unable to mount the normal protective response induced by shear stress in the valve. Generating isogenic NOTCH1+/+ and NOTCH1+/- cell lines using TALEN genome editing identified genes specifically dysregulated due to NOTCH1 heterozygosity rather than differentially expressed due to genetic background and showed rescue of this dysregulation in TALEN-corrected NOTCH1+/+ ECs. We have mapped the gene networks dysregulated in NOTCH1+/- ECs as determined by NOTCH1 ChIP-seq, differentially methylated regions of DNA, and genome-wide differences in the progression of activating and repressive chromatin states that begin to explain the mechanisms by which heterozygosity of a transcription factor leads to disease-specific changes. Determining the consequence of NOTCH1 heterozygous mutations in human patient-specific ECs will reveal novel mechanisms underlying aortic valve disease, leading to potential targets for intervention.

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.

Increased expression of Hes5 protein in Notch signaling pathway in the hippocampus of mice offspring of dams fed a high-fat diet during pregnancy and suckling

Maternal high-fat diet (HFD) impairs hippocampal development of offspring promoting decreased proliferation of neural progenitors, in neuronal differentiation, in dendritic spine density and synaptic plasticity reducing neurogenic capacity. Notch signaling pathway participates in molecular mechanisms of the neurogenesis. The activation of Notch signaling leads to the upregulation of Hes5, which inhibits the proliferation and differentiation of neural progenitors. This study aimed to investigate the Notch/Hes pathway activation in the hippocampus of the offspring of dams fed an HFD. Female Swiss mice were fed a control diet (CD) and an HFD from pre-mating until suckling. The bodyweight and mass of adipose tissue in the mothers and pups were also measured. The mRNA and protein expression of Notch1, Hes5, Mash1, and Delta1 in the hippocampus was assessed by RT-PCR and western blotting, respectively. Dams fed the HFD and their pups had an increased bodyweight and amount of adipose tissue. Furthermore, the offspring of mothers fed the HFD exhibited an increased Hes5 expression in the hippocampus compared with CD offspring. In addition, HFD offspring also expressed increased amounts of Notch1 and Hes5 mRNA, whereas Mash1 expression was decreased. However, the expression of Delta1 did not change significantly. We propose that the overexpression of Hes5, a Notch effector, downregulates the expression of the proneural gene Mash1 in the offspring of obese mothers, delaying cellular differentiation. These results provide further evidence that an offspring's hippocampus is molecularly susceptible to maternal HFD and suggest that Notch1 signaling in this brain region is important for neuronal differentiation.

Expression and significance of DLL4--Notch signaling pathway in the differentiation of human umbilical cord derived mesenchymal stem cells into cardiomyocytes induced by 5-azacytidine.

hUCMSCs were isolated and purified from the umbilical cords of normal or cesarean term deliveries under sterile conditions. Flow cytometry analysis revealed that CD13, CD29, CD44, CD90, and CD105 were highly expressed on the surface of passage-3 hUCMSCs, but negative for CD31, CD34, CD45, and HLA-DR. Immunocytochemistry showed that 5-azacytidine (5-aza) could induce the cTnI expression of hUCMSCs. RT-PCR showed that a stable higher level expression of DLL4 and Notch1 gene in 5-aza-induced group was observed compared to that in the control group. There was a higher expression level in the induced group. Compared with control group, the expression levels of Notch1 were, respectively, increased 6.60, 7.36, 7.595, and 7.805 times at 1, 3, 5, and 7 days after intervention of 5-aza. Statistically higher Ct value of Notch1 mRNA in induced group was observed in comparison with that of the control group (0.51 ± 0.21 vs 7.85 ± 0.35, t = 35.98, P < 0.01). The expression level of DLL4 increased stably compared with the control group. Compared with control group, the expression levels of DLL4 were, respectively, increased 11.53, 10.1, 10.17, and 11.46 times at 1, 3, 5, and 7 days after intervention of 5-aza. There was a significant difference of DLL4 Ct value between the 5-aza-induced group and the control group (1.60 ± 0.49 vs 12.42 ± 0.73, t = 11.71, P < 0.01). In conclusion, hUCMSCs can be differentiated into myocardial cells in vitro. The DLL4-Notch signaling pathway may be involved in the differentiation of hUCMSCs into cardiomyocytes induced by 5-aza.