Clinical significance of notch receptors, NPR3, and DLL-3 in pediatric and adult medulloblastoma patients

Activation of the NOTCH signaling pathway is associated with tumorigenesis. The aim of this study was to investigate NOTCH pathway gene functions and regulatory mechanisms in ovarian cancer (OC). We conducted a bioinformatics analysis of publicly available datasets in order to identify potential NOTCH-related mechanisms, associated genes, biological pathways, and their relation to immune function. Significant differential expression of the NOTCH pathway genes DLL1, DLL3, DLL4, HES1, HEY1, JAG1, NOTCH2, NOTCH3, and NOTCH4 was observed between OC samples and normal controls. Low expression of DLL4 and of NOTCH4 in OC patients was associated with International Federation of Gynecology and Obstetrics (FIGO) stage (p <0.001 and p = 0.036, respectively), while high expression of NOTCH3 was associated with race (p = 0.039) and age (p = 0.044). JAG2 and NOTCH1 expression were significantly associated with progression-free interval (PFI) (p = 0.011 and p = 0.039, respectively). DLL1 (Hazard Ratio (HR): 2.096; 95% CI: 1.522-2.886, p < 0.001) and NOTCH1 (HR: 0.711; 95% CI: 0.514-0.983, p = 0.039) expression were independently associated with PFI in multivariate analysis. DLL1, DLL3, JAG1, JAG2, NOTCH3 and NOTCH4 expression could significantly differentiate OC from non-cancer samples. Genes associated with the NOTCH pathway were mainly enriched in five signaling pathways: the NOTCH signaling pathway, breast cancer, endocrine resistance, Th1 and Th2 cell differentiation, and oxidative phosphorylation. The expression of NOTCH pathway genes was significantly associated with immune cell infiltration. NOTCH pathway genes appear to play an important role in the progression of OC by regulating immune cells, endocrine resistance, Th1 and Th2 cell differentiation, and oxidative phosphorylation. JAG2 and NOTCH1 are potential biomarkers and therapeutic targets for the treatment of OC.

Notch signaling has been suggested to promote the development and maintenance of arteriovenous malformations (AVMs), but whether radiosurgery inhibits Notch signaling pathways in AVMs is unknown. The aim of this study was to examine molecular changes of Notch signaling pathways following radiosurgery and to explore mechanisms of radiosurgical obliteration of "nidus" vessels in a rat model of AVMs. One hundred eleven rats received common carotid artery-to-external jugular vein anastomosis to form an arteriovenous fistula (AVF) model. Six weeks postoperatively, dilated small vessels and capillaries formed a nidus. The rats with AVFs received 25-Gy radiosurgery. The expression of Notch1 and Notch4 receptors and their ligands, Delta-like1 and Delta-like4, Jagged1, Notch downstream gene target HES1, and an apoptotic marker caspase-3 in nidus vessels in the AVF rats was examined immunohistochemically and was quantified using LAS-AF software at 7 time points over a period of 42 days postradiosurgery. The interaction events between Notch1 receptor and Jagged1, as well as Notch4 receptor and Jagged1, were quantified in nidus vessels in the AVF rats using proximity ligation assay at different time points over 42 days postradiosurgery. The expression of Notch1 and Notch4 receptors, Delta-like1, Delta-like4, Jagged1, and HES1 was observed in nidus vessels in the AVF rats pre- and postradiosurgery. Radiosurgery enhanced apoptotic activity (p < 0.05) and inhibited the expression of Notch1 and Notch4 receptors and Jagged1 in the endothelial cells of nidus vessels in the AVF rats at 1, 2, 3, 7, 21, 28, and 42 days postradiosurgery (p < 0.05). Radiosurgery suppressed the interaction events between Notch1 receptor and Jagged1 (p < 0.001) as well as Notch4 receptor and Jagged1 (p < 0.001) in the endothelial cells of nidus vessels in the AVF rats over a period of 42 days postradiosurgery. Radiosurgery induced thrombotic occlusion of nidus vessels in the AVF rats. There was a positive correlation between the percentage of fully obliterated nidus vessels and time after radiosurgery (r = 0.9324, p < 0.001). Radiosurgery inhibits endothelial Notch1 and Notch4 signaling pathways in nidus vessels while inducing thrombotic occlusion of nidus vessels in a rat model of AVMs. The underlying mechanisms of radiosurgery-induced AVM shrinkage could be a combination of suppressing Notch receptor signaling in blood vessel endothelial cells, leading to a reduction in nidus vessel size and thrombotic occlusion of nidus vessels.

Objective To explore whether hypertonic saline would partake in regulating Notch signaling in microglia in experimentally induced cerebral ischemic rats. Methods Male SD rats were randomly divided into sham group, cerebral ischemia group, normal saline group (NS group), 10% hypertonic saline group (10%HS group), the model of cerebral ischemia were established in all rats except the sham group by using middle cerebral artery occlusion (MCAO). After 2 hours of MCAO, the rats were through reperfusion for 24 h. In addition, rats in the normal saline group and 10% HS group were respectively treated with a continuous intravenous injection of normal saline (0.3 mL/h) and 10% HS (0.3 mL/h) by tail vein for 24 h. Immunofluorescence methods, RT-PCR and Western blot were used to detect the expression of Notch1 and intracellular Notch receptor domain (NICD). All data was analyzed by one-way analysis of variance (ANOVA), The intergroup comparisons were analyzed by the least-significant-difference (LSD) tests. Differences were considered statistically significant if P<0.05. Results Immunofluorescence showed that the expression of Notch1 and NICD were significantly increased in the microglia around peri-ischemia area in cerebral ischemia group and normal saline group compared to sham group; the expression of Notch1 and NICD in the microglia around peri-ischemia area were significantly reduced in 10% HS group compared to ischemia group and NS group. RT-PCR showed that the mRNA expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group ( sham group: 1.000±0.076; ischemia group: 2.203±0.283; NS group: 1.616±0.185; P<0.01); however, it was significantly reduced in 10% HS group compared to ischemia group and NS group ( ischemia group: 2.203±0.283; NS group: 1.616±0.185; 10%HS group: 1.202±0.177; P<0.05). Western blot showed that the protein expression of Notch1 was significantly increased in ischemia group and NS group compared to sham group ( sham group: 0.290±0.079; ischemia group: 0.750±0.029; NS group: 0.765±0.182; P<0.01); but was significantly reduced in 10% HS group compared to ischemia group and NS group (ischemia group: 0.750±0.029; NS group: 0.765±0.182; 10%HS group: 0.390±0.195; P<0.05). The protein expression of NICD was significantly increased in ischemia group and NS group compared to sham group ( sham group: 0.401±0.196; ischemia group: 0.906±0.359; NS group: 0.847±0.153; P<0.01); but was significantly reduced in 10% HS group compared to ischemia group and NS group ( ischemia group: 0.906±0.359; NS group: 0.847±0.153; 10%HS group: 0.561±0.165; P<0.05). Conclusion Our results suggest that HS markedly suppresses Notch signaling in microglia around the ischemia tissue area in experimental induced cerebral ischemic rats. Key words: Hypertonic saline; Normal saline; Cerebral ischemia; Middle cerebral artery occlusion (MCAO); Microglia; Notch signaling; Notch1; Notch receptor domain (NICD)

MS05.03 Notch Signalling

The Notch signaling pathway, via cell‐cell communication, is critical during normal vascular development and homeostasis, and it is known to regulate vascular smooth muscle (VSMC) phenotype, including proliferation; however, few studies have addressed the role of Notch signaling in the mature vasculature. The current study tested the hypothesis that Notch signaling is present and active at mature myoendothelial junctions (MEJs), signaling hubs that occur at breaks in the internal elastic lamina (IEL). In mature, intact normal adult mouse coronary resistance microvessels (CRMs) embedded within heart sections, we observed Notch1, Notch2, Notch3, and Jagged1 expression within the fenestrae of the CRM IEL where MEJs are known to occur. Immunofluorescence clearly shows bridging from the VSMCs to endothelial cells (ECs). Immunofluorescence in larger mouse left anterior descending (LAD) coronary arteries demonstrate the expression Notch2 and Notch3 protein immunofluorescence within fenestrae of the IEL. We found that, on average, 39% and 33% of IEL fenestrae were positive for Notch2 and Notch3 expression, respectively. We also confirmed the presence of Notch3 expression in the MEJ by immunogold electron microscopy. In normal mouse femoral arteries, we found that, on average, 46%, 51%, and 58% of IEL fenestrae were positive for Notch1, Notch3, and Jagged1 expression, respectively. To determine whether Notch signaling components were localized to the MEJ, we co‐cultured primary human coronary ECs (hcECs) and primary human coronary VSMCs (hcVSMCs) across transwell inserts, and we utilized fluorescent microscopy to immunostain for Pai‐1, Jagged1, Notch1, and Notch2. PAI‐1 immunofluorescence was observed on the EC side of the transwells and within the transwell pores, indicating the formation of in vitro MEJs. We also identified distinct staining of Notch1, Notch2, and Jagged1 in the pores of the co‐culture transwells, which co‐localized with PAI‐1 within the pores. To assess whether Notch signaling was active at the MEJ, we co‐cultured hcECs and hcVSMCs on transwells to create in vitro MEJs. Smooth muscle cells were first transfected with a Notch‐sensor luciferase plasmid followed by transwell co‐culture of the transfected cells with endothelial cells. Notch activity was stimulated by 3‐fold across the MEJ in hcVSMC (1.00 ± 0.15 vs. 3.02 ± 0.43, p&lt;0.001), which was completely abrogated by the non‐selective Notch γ‐secretase inhibitor, DAPT (1.15 ± 0.16, p&lt;0.01). In this study, we demonstrate for the first time that Notch receptors and ligands are expressed within and are active at mature coronary MEJs, demonstrating a previously unrecognized mode of Notch signaling regulation between the endothelium and smooth muscle.Support or Funding InformationFunding: EW was supported by NIH R25HD086885. NIH R00HL116769, R21EB026518, and S10OD023438 to AJT.

Since the Notch signaling pathway plays a vital role in cell proliferation, oridonin restrains cancer cell growth bylimiting the Notch pathway. The study aims to investigate whether oridonin can kill normal epithelial cells similar tocancer cells in vitro. Also, the study aims to find whether the inhibition of Notch-1 by oridonin is on a transcriptionallevel. Methods: The study will use human epithelial cells (MCF10A) and breast cancer cells (MCF7). These two typesof cells will be treated with various concentrations of oridonin (0.01mM, 0.1mM, 1mM, and 10mM) for a differentamount of time (24h, 48h, 72h). The cell viability will be measured by WST-1 assay. Possible Results: There areeight possible results, three for the proliferation of epithelial cells and two for the transcriptional level of Notch-1 inepithelial cells. Normal epithelial cells experience a more significant loss of viable cells compared with cancer cells:(1) with decreased transcription and expression of Notch-1; (2) with decreased expression of Notch-1 but no decreasein transcription; (3) with a decrease in transcription but no decrease in expression of Notch-1 (4) with no decreasein expression and transcription of Notch-1. Normal epithelial cells experience a similar or less loss of viable cellscompared with cancer cells, (5) with decreased transcription and expression of Notch-1; (6) with decreased expressionof Notch-1 but no decrease in transcription; (7) with a decrease in transcription but no decrease in expression of Notch-1(8) with no decrease in expression and transcription of Notch-1. Conclusion: The loss of normal cells is considered atrade-off for the anti-tumor activity of oridonin. If little or no damage to non-tumorigenic epithelial cells is detected,then oridonin may be a good cure for breast cancer with fewer side effects. Also, by investigating whether the inhibitionof Notch-1 by oridonin is on a transcriptional level, we can gain more insights into the mechanism of inhibition.

ObjectivesTo explore the expression of Notch signal and cytokines by oxidised low-density lipoprotein (ox-LDL) in macrophages of human acute monocytic leukaemia cell line (THP1) and to search for possible mechanism...

Adult medulloblastoma is a rare tumor with few retrospective studies published so far. The role of adjuvant chemotherapy or chemotherapy at relapse is unclear. This study reports therapy and outcome in all adult (>or=16 years old) medulloblastoma (n=34) and supratentorial primitive neuroectodermal tumor (PNET) patients (n=2) treated in 2 neuro-oncological centers between 1976 and 2002. The median age was 24.5 years (range 16-76). After resection, 16 patients were treated with craniospinal radiotherapy alone, 20 patients also received adjuvant chemotherapy (8 vincristine, CCNU, cisplatin; 7 methotrexate alone or methotrexate/vincristine-based polychemotherapy; 5 other protocols). Median survival in the whole cohort was 126 months (2+ - 200+months). Five-year and 10-year survival rates were 79 % and 56%. Adjuvant chemotherapy was associated with a non-significant trend to prolonged survival (relative risk (RR) 1.89; p=0.068). The median progression-free survival (PFS) after primary therapy was 83 months. At relapse, 10 of 12 evaluable patients achieved a complete response upon second-line therapy. The median survival times from first (n=17) and second relapse (n=9) were 21 months (0-67+ months; 5/17 without second relapse) and 20 months (1-29 months). Cox regression analysis revealed the infiltration of the floor of the 4(th) ventricle at diagnosis as the only therapy-independent prognostic factor (RR 0.48; p=0.03). In conclusion, adjuvant chemotherapy may prolong survival in adult medulloblastoma patients. Moreover, second-line therapy may be beneficial for these patients. As in pediatric medulloblastoma patients, primary infiltration of the floor of the 4(th) ventricle indicates a poor prognosis.

The interactions between Notch (N) receptors and their transmembrane ligands, Jagged1 (JI) and Delta1 (Dl1), mediate signaling events between neighboring cells that are crucial during embryonal development and in adults. Since the non-transmembrane extracellular form of J1 acts as an antagonist of N activation in NIH 3T3 mouse fibroblast cells and induces fibroblast growth factor 1 (FGF1)-dependent transformation (Small, D., Kovalenko, D., Soldi, R., Mandinova, A., Kolev, V., Trifonova, R., Bagala, C., Kacer, D., Battelli, C., Liaw, L., Prudovsky, I., and Maciag, T. (2003) J. Biol. Chem. 278, 16405-16413), we examined the potential redundant functions of the two subfamilies of Notch ligands and report that while the soluble (s) forms of both Dl1 and J1 act as N signaling antagonists in NIH 3T3 cells, they do display disparate functions. While sJ1 induced an attenuation of cell motility which is accompanied by a decrease in actin stress fibers and an increase in adherence junctions, sDl1 does not. However, sJ1, like sDl1, induces a NIH 3T3 cell tranformed phenotype mediated by FGF signaling. Because the inhibition of N signaling by sJ1 and sDl1 is rescued by dominant-negative Src expression, we suggest that there may be cooperation between the Notch and Src signaling pathways.

The aim of this study was to investigate the influence of micro-ribonucleic acid-34a (miR-34a) on preeclampsia through the Notch signaling pathway. The expressions of miR-34a, Notch-1, Notch-2, and Notch-3 in the placenta of 39 preeclampsia patients and 42 normal patients were detected by immunohistochemistry and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The correlations between miR-34a expression with the expressions of Notch-1, Notch-2 and Notch-3 were analyzed, respectively. Besides, placental trophoblasts were isolated from preeclampsia patients and cultured in vitro. The expressions of miR-34a, Notch-1, Notch-2 and Notch-3 in placental trophoblasts were analyzed. Furthermore, the influences of miR-34a on the protein expressions of Notch-1, Notch-2, Notch-3, and hairy and enhancer of split-1 (Hes-1) in the Notch signaling pathway were analyzed by Luciferase reporter gene assay and Western blotting. The role of Notch in trophoblast invasion was investigated through the Notch inhibitors. In addition, its influence on the expression of urokinase-type plasminogen activator (uPA) was studied by miR-34a overexpression. The expressions of miR-34a and Notch-1 were correlated with preeclampsia in the placentas of preeclampsia patients and normal patients to a certain degree. The expression of miR-34a in preeclamptic placenta was significantly higher than that of the normal placenta (p<0.05). However, Notch-1 expression was markedly lower in preeclamptic placenta (p<0.05). No significant differences were found in the expressions of Notch-2 and Notch-3 between the two types of placentas (p>0.05). MiR-34a had a remarkable negative correlation with Notch-1 expression in the Notch family (p<0.001, r=-0.5775). RT-PCR results revealed that the mRNA expression of miR-34a in placental trophoblasts of patients with preeclampsia was notably higher than that of normal people (p<0.01). However, Western blotting demonstrated that the protein expressions of Notch-1, Notch-2 and Notch-3 exhibited the opposite results. Additionally, the protein expression of Notch-1, Notch-2, Notch-3 and Hes-1 in trophoblasts transfected with pre-miR-34a was significantly decreased. The treatment with Notch inhibitors markedly reduced the trophoblast invasion. Furthermore, miR-34a overexpression or intracellular domain of Notch (ICN) overexpression regulated uPA expression. MiR-34a regulates uPA system through the Notch signal transduction, thereby regulating the invasion of placental trophoblasts in patients with preeclampsia.

IntroductionThe Notch signaling pathway is involved in the development of many diseases; it regulates the development of dendritic cells (DCs), and affects the immune response of DC-mediated T cells. We previously found that ferritin and malate dehydrogenase (mMDH) in Echinococcus granulosus (E.granulosus) induced different immune responses through sensitized DCs. Therefore, in the study we explored whether the Notch signaling pathway affects the development and differentiation of DCs, causing changes in the immune response of DCs sensitized with E. granulosus antigens, and clarified whether it is involved in E.granulosus infection. MethodsWe used the Notch signaling pathway inhibitor [N-[3,5-difluorophenace-tyl] -L-alanyl]-S-phenylglycinet-butyl ester (DAPT) or activator Jagged1 to construct in vitro cell models with blocked or activated Notch signaling respectively. We analyzed the effect of Notch signaling on the development and differentiation of DCs by detecting their morphology, migration function, capacity to promote T cell proliferation, and cytokine secretion. We observed the changes in DC response to E. granulosus antigens and the mediated immune response. ResultsDAPT inhibited the development and maturation of DCs, which were in a non-responsive or incompetent state, reduced the sensitization of DCs to Eg.ferritin, weakened the migration ability of DCs, disrupted their ability to mediate T-cell proliferation, reduced DC expression of MHCII, CD80, CD60, and CD40 co-stimulatory molecules, prevented the secretion of cytokines and attenuated the expression of Notch1, Notch2, Notch3 receptors, Jagged1, Delta-like 4 (Delta4), and Hes1. Following Jagged1 addition, the function of DCs was restored to some extent, and the expression of Notch1, Delta4 and Hes1 was activated in response to the stimulation of Eg.ferritin. However, Eg.mMDH stimulated DCs to produce an immune response showing weak interference by DAPT and Jagged1.DiscussionThe study suggests that the Notc h signaling pathway is involved in the Eg.ferritin-sensitized DC-mediated immune response, which may become a new target for treating E.granulosus infection.

e16139 Background: Despite the recent addition of atezolizumab to advanced HCC first-line treatment, response biomarkers are not well studied. The involvement of the NOTCH gene family in HCC tumorigenesis has been previously discussed, yet insights on its effect on the immune microenvironment are still lacking. Methods: We systematically analyzed differentially expressed genes (DEGs) in the KEGG NOTCH pathway across HCC transcriptomics datasets from the HCCDB platform. Immune and prognostic analyses were done using The Cancer Genomic Atlas (TCGA) HCC cohort (n = 372). The TIMER 2.0 tool was used to assess immune infiltration. cBioportal was used for tumor mutational burden (TMB) and microsatellite instability (MSI) correlations. The TIDE tool assessed T cell dysfunction and DEGs efficacy predictive value using the only trial administering atezolizumab (IMvigor210, n = 348). Results: Four DEGs were identified: NCSTN, APH1A, NOTCH3, HDAC2. All four genes were significantly directly associated (spearman’s ρ &gt; 0.1, P &lt; 0.05) with immunosuppressive cells: Tregs, macrophage M2 cells, cancer-associated fibroblasts (CAF), and myeloid-derived suppressor cells (MDSC). HDAC2 was associated with higher T cell dysfunction (z score = 2.07, P = 0.04). High NOTCH3 and low HDAC2 expression served as favorable overall survival (OS) prognostic markers (HR: 0.66, 95% CI: 0.48 - 0.90, P = 0.008 &amp; HR: 1.52, 95% CI: 1.12–2.07, P = 0.006, respectively). Stratification based on the expression of these two genes and strongly associated immunosuppressive cells’ infiltration levels (ρ &gt; 0.5), revealed two unique patients’ subsets with worse OS marked by “low NOTCH3 expression with high CAF levels” and “high HDAC2 expression with high MDSC levels”. Regarding immune checkpoint genes expression, NOTCH3 and HDAC2 expression levels were directly correlated with CTLA-4, PD-1, and TIM-3, whereas NCSTN and APH1A were inversely related with PD-L1, LAG-3, and TIM-3 after correction for multiple hypothesis testing (ρ &gt; 0.2, Q value &lt; 0.05). TMB count was inversely associated with NOTCH3 expression (ρ = - 0.2, P &lt; 0.001), as for the MSI sensor score, NOTCH3 (ρ = -0.29) and APH1A (ρ = 0.29) expression had inverse and direct significant associations, respectively (P &lt; 0.0001). Exploratory subgroup analysis from the IMvigor210 trial on metastatic urothelial carcinoma patients on atezolizumab showed a significant difference in OS favoring low NOTCH3 expression (P = 0.003) and high APH1A expression (P = 0.049). Conclusions: DEGs from the NOTCH family seem to impose an immunosuppressive effect on HCC’s microenvironment. High NOTCH3 and low HDAC2 expression were identified as potential favorable prognostic markers. Interestingly, high NOTCH3 expression translated into worse OS in patients on anti-PD-L1 which could be owed to the inverse relation with TMB and MSI described in our study, thus deserving further translational investigations.

Introduction: Triple negative breast cancer (TNBC) is a particularly aggressive form of the disease whose incidence is disproportionate in obese African American and Latina women. TNBC is commonly associated with acute early onset, poor survival and acquired drug-resistance. Obesity, characterized by high levels of leptin, is frequently found among these ethnic groups. We hypothesize that intact leptin-Notch-Wnt signaling crosstalk affects the development of drug-resistance in TNBC. Objective: To determine whether leptin signaling induces Notch/Wnt pathway crosstalk, decreasing the effectiveness of cisplatin in TNBC cells. Materials &amp; Methods: TNBC cells (MDA MB 231 and HCC1806) and ER+ MCF-7 (as control) were treated with pharmacological doses of leptin and agonists of the Notch and Wnt pathways JAG1-17 mer peptide and Wnt-1, respectively. In addition, the cells were challenged with the leptin receptor, Notch pathway, and Wnt pathway antagonists LPrA2, DAPT, and Wnt-1 respectively. The impact of these molecules on cisplatin anti-cancer effects was also determined. Activation of Wnt (total/pβ-catenin), expression of Notch (Notch 1-4 and JAG1/Dll-4 and targets: survivin/Hey2), cell proliferation and apoptosis were determined by Western blot and flow cytometry. β-catenin levels were also investigated by immunofluorescence. We also determined the impact of the inhibition of leptin signaling (via LPrA2) on the expression of Wnt and Notch in DMBA-induced breast cancer from DIO (diet-induced obese) mice. Results: We found that leptin increased the levels of β-catenin mainly in TNBC cells. Similarly, inhibition of leptin signaling decreased Wnt/Notch expression in DMBA/DIO-breast cancer. Interestingly, leptin increased expression of Notch and attenuated the detrimental effects of cisplatin on breast cancer cells. Wnt-1 affected Notch levels but its effects were less pronounced. Conclusions: Leptin is able to induce the Notch and Wnt signaling pathways in breast cancer. Cisplatin effects were attenuated by leptin in TNBC probably through its crosstalk with Wnt and Notch signaling pathways. These results are particularly relevant for TNBC and obese patients, and suggest that leptin inhibition could be useful in increasing the chemotherapeutic effectiveness in TBNC by disrupting leptin-Wnt-Notch crosstalk. [This work was supported in part by NIH/NCI1SC1CA138658-02; NIH/ARRA/3SC1CA138658-02S1 and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP); CREDO (MSCR) 2R25RR017694-06A1 (to L.S.C); NIH/NIGMS R25 GM058268 and NCRR 5P20RR11104 (to T.Z.M); and facilities and support services at MSM (NIH RR03034 and 1C06 RR18386)]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 785. doi:1538-7445.AM2012-785

In cancer, DNA methylation causes alteration in gene expression affecting important signal transduction pathways. Since methylation occurs at a very early stage and is reversible, hypermethylated promoters hold great promise as biomarkers for early detection and effective drug targets for gene reactivation. The Notch signaling pathway is one such important developmental pathway governing cell fate decisions. Dysregulated Notch signaling is found to have a prominent role in the development of various cancers. Glioblastoma (GBM) is the most common primary brain tumor, with a very poor prognosis despite advanced treatment facilities due to poor molecular stratification. It is therefore important to study genetic and epigenetic events leading to gliomagenesis and to guide new treatment strategies. The aim of this study was to detect Notch pathway genes potentially regulated by promoter methylation in human GBM. We used real-time PCR and quantitative methylation-specific PCR to study methylation status of underexpressed Notch pathway genes from human GBM formalin-fixed, paraffin-embedded sections. 50% of samples showed methylation at Notch4 gene promoter at levels higher than 25 % (P &amp;lt; 0.05). However, JAG2 gene promoter was estimated to be methylated at a negligible level (&amp;lt;2 %) (P &amp;lt; 0.05). Our study showed poor gene expression and high-level methylation of Notch4 gene, suggesting a possibility of epigenetic silencing. However, low JAG2 gene expression despite not being highly methylated suggests lack of correlation between gene expression and promoter methylation and the presence of alternative regulatory mechanisms. This study for the first time provides gene expression and DNA methylation profiles of Notch pathway genes from GBM patient samples. We have identified genes whose expression may be regulated by epigenetic mechanisms and thus can be used as potential biomarkers. This study will eventually give clues to improve diagnosis, prognosis, and treatment of GBM. Citation Format: Madhuri G S Aithal, Rajeswari Narayanappa. Role of promoter methylation in regulation of Notch4 and JAG2 gene expression in human glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A177.

Notch and transforming growth factor-beta (TGFbeta) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFbeta1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle alpha-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFbeta1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-alpha (SM22alpha) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFbeta1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFbeta1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFbeta1 treatment led to synergistic activation of a TGFbeta-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22alpha promoters. Thus, Notch and TGFbeta coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.