Expression Of Notch Pathway Genes Research Articles

NOTCH Pathway Genes in Ovarian Cancer: Clinical Significance and Associations with Immune Cell Infiltration.

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.

Pan-cancer molecular subtypes of metastasis reveal distinct and evolving transcriptional programs

Molecular mechanisms underlying cancer metastasis span diverse tissues of origin. Here, we synthesize and collate the transcriptomes of patient-derived xenografts and patient tumor metastases, and these data collectively represent 38 studies and over 3,000 patients and 4,000 tumors. We identify four expression-based subtypes of metastasis transcending tumor lineage. The first subtype has extensive copy alterations, higher expression of MYC transcriptional targets and DNA repair genes, and bromodomain inhibitor response association. The second subtype has higher expression of genes involving metabolism and prostaglandin synthesis and regulation. The third subtype has evidence of neuronal differentiation, higher expression of DNA and histone methylation genes and EZH2 transcriptional targets, and BCL2 inhibitor response association. The fourth subtype has higher expression of immune checkpoint and Notch pathway genes. The metastasis subtypes reflect expression differences from paired primaries, with subtype switching being common. These subtypes facilitate understanding of the molecular underpinnings of metastases beyond tissue-oriented domains, with therapeutic implications.

Expression analysis elucidates the roles of Nicastrin, Notch4, and Hes1 in prognosis and endocrine-therapy resistance in ER-positive breast cancer patients.

Although some proposed mechanisms responsible for tamoxifen resistance have already been present, further study is needed to determine the mechanisms underlying tamoxifen resistance more clearly. The critical role of Notch signaling has been described in promoting resistance in therapeutics, but there is little information about its role in tamoxifen resistance progression. In the present study, the expression of Notch pathway genes, including Notch4, nicastrin and the Notch downstream target Hes1 was evaluated using quantitative RT-PCR in 36 tamoxifen-resistant (TAM-R) and 36 tamoxifen-sensitive (TAM-S) patients. Expression data were correlated with the clinical outcome and survival of patients. mRNA levels of Notch4 (fold change = 2.7), nicastrin (fold change = 6.71), and Hes1 (fold change= 7.07) were significantly higher in TAM-R breast carcinoma patients compared to sensitive cases. We confirmed all these genes were co-expressed. Hence, it seems that Notch signaling is involved in tamoxifen resistance in our TAM-R patients. Obtained results showed that Hes1, nicastrin, and Notch4 mRNA upregulation was correlated with the N stage. The extracapsular nodal extension was associated with nicastrin and Notch4 overexpression. Moreover, nicastrin overexpression was correlated with perineural invasion. Hes1 upregulation was also associated with nipple involvement. Finally, the Cox regression proportional hazard test revealed that overexpression of nicastrin was an independent worse survival factor. Presumably, upregulation of the Notch pathway may be involved in tamoxifen resistance in breast cancer patients.

Notch signaling as a novel target for uterine leiomyosarcoma (259)

<b>Objectives:</b> The Notch signaling pathway is evolutionarily conserved and has been implicated as a potential therapeutic target in several cancers. Uterine leiomyosarcoma (uLMS) is a rare and aggressive cancer with poor survival, higher prevalence in Black women than White women, and few effective adjuvant therapies. The objective of this study was to determine the expression of Notch in uLMS as a foundation for investigating the Notch signaling pathway as a potential therapeutic target in uLMS. <b>Methods:</b> We analyzed previously published mRNA sequencing (seq) datasets on LMS, including uLMS and other sarcoma patient tissue samples for expression of Notch pathway genes. We performed immunostaining for <i>NOTCH1</i> and <i>NOTCH3</i> on uterine tissue from benign hysterectomies (BH, <i>n</i>=3) and uLMS samples (<i>n</i>=30) from a multiple organ LMS tissue array (US Biomax, Inc). <i>NOTCH1</i> expression was measured as a percent positive stain per area, and <i>NOTCH3</i> expression was quantified with an H-Score. We analyzed Notch intracellular domain (ICD) protein expression in human uterine smooth muscle cell lines (hUT-SMC) and uLMS cell lines, SK-UT-1B and SK-LMS-1, by Western blot, and expression of Notch effectors, <i>HES1, HEY1,</i> and <i>NRARP,</i> by qPCR and immunofluorescence. Numerical data were reported as mean ± SEM or median ± interquartile range and compared using unpaired t-test or Mann-Whitney U, with p≤0.05 being statistically significant. <b>Results:</b> mRNA seq analysis revealed similar levels of expression for <i>NOTCH1, 2,</i> and <i>3</i> and <i>HES1</i> in LMS compared to other sarcomas. <i>NOTCH4</i> was not included in the original mRNA seq data and thus could not be evaluated for expression levels. Immunostaining revealed that median H-score of <i>NOTCH3</i> in uLMS was higher than in BH (211.4, IQR: 165.6-239.3 vs 100.3, IQR: 100.0-100.5, p≤0.05), whereas mean expression of <i>NOTCH1</i> was similar (3.7 ± 1.6% vs 1.9 ± 0.5%, p>0.05). Western blot analysis demonstrated that ICD for <i>NOTCH1-4</i> was expressed in hUT-SMC, N1ICD and N2ICD were expressed in SK-UT-1B, and N1ICD, N2ICD, and N4ICD were expressed in SK-LMS-1. mRNA expression of <i>HES1, HEY1,</i> and <i>NRARP</i> and nuclear expression of <i>HES1</i> indicate active Notch signaling in SK-LMS-1 and SK-UT-1B cells. <b>Conclusions:</b> Notch is expressed in uLMS patient tissues and uLMS cell lines. Notch signaling and distinct expression of Notch proteins in uLMS cell lines support the investigation of Notch inhibition as a potential target for treatment options in uLMS. Further studies are warranted to determine the efficacy of targeting Notch inhibition using therapies, such as gamma-secretase inhibitors alone or in combination with a cytotoxic agent.

Magnesium Ions Promote In Vitro Rat Bone Marrow Stromal Cell Angiogenesis Through Notch Signaling.

Bone defects are often caused by trauma or surgery and can lead to delayed healing or even bone nonunion, thereby resulting in impaired function of the damaged site. Magnesium ions and related metallic materials play a crucial role in repairing bone defects, but the mechanism remains unclear. In this study, we induced the angiogenic differentiation of bone marrow stromal cells (BMSCs) with different concentrations of magnesium ions. The mechanism was investigated using γ-secretase inhibitor (DAPT) at different time points (7 and 14days). Angiogenesis, differentiation, migration, and chemotaxis were detected using the tube formation assay, wound-healing assay, and Transwell assay. Besides, we analyzed mRNA expression and the angiogenesis-related protein levels of genes by RT-qPCR and western blot. We discovered that compared with other concentrations, the 5mM magnesium ion concentration was more conducive to forming tubes. Additionally, hypoxia-inducible factor 1 alpha (Hif-1α) and endothelial nitric oxide (eNOS) expression both increased (p < 0.05). After 7 and 14days of induction, 5mM magnesium ion group tube formation, migration, and chemotaxis were enhanced, and the expression of Notch pathway genes increased. Moreover, expression of the Notch target genes hairy and enhancer of split 1 (Hes1) and Hes5 (hairy and enhancer of split 5), as well as the angiogenesis-related genes Hif-1α and eNOS, were enhanced (p < 0.05). However, thesetrendsdid not occur when DAPT was applied. This indicates that 5mM magnesium ion is the optimal concentration for promoting the angiogenesis and differentiation of BMSCs in vitro. By activating the Notch signaling pathway, magnesium ions up-regulate the downstream genes Hes1 and Hes5 and the angiogenesis-related genes Hif-1α and eNOS, thereby promoting the angiogenesis differentiation of BMSCs. Additionally, magnesium ion-induced differentiation enhances the migration and chemotaxis of BMSCs. Thus, we can conclude that magnesium ions and related metallic materials promote angiogenesis to repair bone defects. This provides the rationale for developing artificial magnesium-containing bone materials through tissue engineering.

Study on the difference of osteogenesis and Notch signaling pathway expression in biphasic calcium-phosphorus ceramic granule materials with different microstructure.

Different microstructures including micropore diameter, micropore volume, and micropore area of biphasic calcium phosphate (BCP, hydroxyapatite: β-tricalcium phosphate=8:2) ceramics granules were obtained by varying their sintering temperatures. Sprague-Dawley rat bone marrow-derived stem cells (BMSCs) were co-cultured with BCPs in vitro study and the BMSCs showed different degrees of proliferative activity under the influence of three materials. Cell proliferation and vitality were assessed. Three kinds of BCPs were implanted in the dorsal muscle of beagle dogs. At 1, 2, and 3 months, histological analyses were conducted to estimate the rate of osteogenesis. Expression of Notch pathway genes and osteogenic-related genes were detected by quantitative real-time polymerase chain reaction (q-rtPCR). The proportion of osteogenesis area increased to:48.75 ± 4.20%, 29.48 ± 1.55%, and 26.58 ± 3.86% at 3 months after the implantation (1050, 1150, 1250). Significant differences were observed in the upregulation of Notch pathway genes among different BCPs. BCPs with different micropore diameters have different ectopic osteogenesis effects and led to up-regulation of the Notch signaling pathway genes to different extents.

Prognostic Signatures and Therapeutic Value Based on the Notch Pathway in Renal Clear Cell Carcinoma.

The Notch family of genes encodes a group of highly conserved cell surface membrane receptors, which are involved in one of the key pathways that determine cell growth, differentiation, and apoptosis in embryonic tissues. Furthermore, abnormal expression of Notch genes is closely related to the occurrence and development of several cancers. To date, no specific treatment of RCC has been reported to relate to the Notch pathway. Therefore, we detected Notch pathway genes in series of tumors, as well as potential compounds targeting the Notch pathway, with a focus on the mechanism of Notch pathway action in kidney renal clear cell carcinoma (KIRC). Samples from KIRC patients were divided into three clusters based on the mRNA expression of Notch pathway genes. In addition, we investigated the potential targets of the Notch pathway, predicted the IC50 of several classical targeted therapies, and analyzed their correlation with the Notch pathway. Finally, LASSO regression analysis was performed to build a model to predict survival in KIRC patients. These results suggest that therapies targeting the Notch pathway could be more efficiently studied based on the Notch score and that we can predict the prognosis of patients with KIRC based on the expression of Notch pathway genes. Most importantly, these results may provide a solid theoretical basis for future research on therapeutic targets for patients with KIRC.

Endogenous expression of Notch pathway molecules in human trabecular meshwork cells

PurposeCells in the trabecular meshwork sense and respond to a myriad of physical forces through a process known as mechanotransduction. Whilst the effect of substratum stiffness or stretch on TM cells have been investigated in the context of transforming growth factor (TGF-β), Wnt and YAP/TAZ pathways, the role of Notch signaling, an evolutionarily conserved pathway, recently implicated in mechanotransduction, has not been investigated in trabecular meshwork (TM) cells. Here, we compare the endogenous expression of Notch pathway molecules in TM cells from glaucomatous and non-glaucomatous donors, segmental flow regions, and when subjected to cyclical strain, or grown on hydrogels of varying rigidity. MethodsPrimary TM from glaucomatous (GTM), non-glaucomatous (NTM) donors, and from segmental flow regions [high flow (HF), low flow (LF)], were utilized between passages 2–6. Cells were (i) plated on tissue culture plastic, (ii) subjected to cyclical strain (6 h and 24 h), or (iii) cultured on 3 kPa and 80 kPa hydrogels. mRNA levels of Notch receptors/ligands/effectors in the TM cells was determined by qRT-PCR. Phagocytosis was determined as a function of substratum stiffness in NTM-HF/LF cells in the presence or absence of 100 nM Dexamethasone treatment. ResultsInnate expression of Notch pathway genes were significantly overexpressed in GTM cells with no discernible differences observed between HF/LF cells in either NTM or GTM cells cultured on plastic substrates. With 6 h of cyclical strain, a subset of Notch pathway genes presented with altered expression. Expression of Notch receptors/ligands/receptors/inhibitors progressively declined with increasing stiffness and this correlated with phagocytic ability of NTM cells. Dexamethasone treatment decreased phagocytosis regardless of stiffness or cells isolated from segmental outflow regions. ConclusionsWe demonstrate here that the Notch expression in cultured TM cells differ intrinsically between GTM vs NTM, and by substratum cues (cyclical strain and stiffness). Of import, the most apparent differences in gene expression were observed as a function of substratum stiffness which closely followed phagocytic ability of cells. Interestingly, on soft substrates (mimicking normal TM stiffness) Notch expression and phagocytosis was highest, while both expression and phagocytosis was significantly lower on stiffer substrates (mimicking glaucomatous stiffness) regardless of DEX treatment. Such context dependent changes suggest Notch pathway may play differing roles in disease vs homeostasis. Studies focused on understanding the mechanistic role of Notch (if any) in outflow homeostasis are thus warranted.

TWIST1 correlates with Notch signaling pathway to develop esophageal squamous cell carcinoma.

Notch signaling pathway mediates different biological processes including stem cell self-renewal, progenitor cell fate decision, and terminal differentiation. TWIST1 plays a key role in tumor development and metastasis through inducing epithelial-mesenchymal transition (EMT). Expression of the core transcriptional complex of Notch pathway and its target genes, as well as TWIST1 overexpression, are closely related to the aggressive clinicopathological variables of esophageal squamous cell carcinoma (ESCC). Here we aimed to functionally elucidate probable crosstalk between TWIST1 and Notch pathway in ESCCs. Correlation between TWIST1 and Notch target genes was analyzed in 50 ESCCs and corresponding normal tissues. Using retroviral system, enforced expression of TWIST1 was established in ESCC line KYSE-30 cells and expression of Notch signaling genes was assessed. Significant correlation between TWIST1 and HEY1/HEY2 expression was found in different pathological variable of ESCC poor prognosis. Induced expression of TWIST1 in KYSE-30 cells caused a noteworthy increase of Notch pathway genes expression revealing regulatory role of TWIST1 on Notch signaling genes in the cells. Based on existed correlations between expression of TWIST1 and Notch pathway genes in different pathological features of ESCC patients, as well as KYSE-30 cell line, we may extrapolate that TWIST1 is involved in aggressiveness of the disease through regulation of Notch signaling genes. To the best of knowledge, this is the first report describing the impact of TWIST1 on Notch cascade genes in ESCC.

Notch pathway up-regulation via curcumin mitigates bisphenol-A (BPA) induced alterations in hippocampal oligodendrogenesis

CNS myelination process involves proliferation and differentiation of oligodendrocyte progenitor cells (OPCs). Defective myelination causes onset of neurological disorders. Bisphenol-A (BPA), a component of plastic items, exerts adverse effects on human health. Our previous studies indicated that BPA impairs neurogenesis and myelination process stimulating cognitive dysfunctions. But, the underlying mechanism(s) of BPA induced de-myelination and probable neuroprotection by curcumin remains elusive. We found that curcumin protected BPA mediated adverse effects on oligosphere growth kinetics. Curcumin significantly improved proliferation and differentiation of OPCs upon BPA exposure both in-vitro and in-vivo. Curcumin enhanced the mRNA expression and protein levels of myelination markers in BPA treated rat hippocampus. Curcumin improved myelination potential via increasing β-III tubulin-/MBP+ cells (neuron-oligodendrocyte co-culture) and augmented fluoromyelin intensity and neurofilament/MBP+ neurons in vivo. In silico docking studies suggested Notch pathway genes (Notch-1, Hes-1 and Mib-1) as potential targets of BPA and curcumin. Curcumin reversed BPA mediated myelination inhibition via increasing the Notch pathway gene expression. Genetic and pharmacological Notch pathway inhibition by DAPT and Notch-1 siRNA exhibited decreased curcumin mediated neuroprotection. Curcumin improved BPA mediated myelin sheath degeneration and neurobehavioral impairments. Altogether, results suggest that curcumin protected BPA induced de-myelination and behavioural deficits through Notch pathway activation.

The Notch Pathway Promotes Osteosarcoma Progression through Activation of Ephrin Reverse Signaling.

Despite significant advancements in the diagnosis and treatment of osteosarcoma, the molecular mechanisms underpinning disease progression remain unclear. This work presents strong clinical and experimental evidence demonstrating that Notch signaling contributes to osteosarcoma progression. First, using a cohort of 12 patients, Notch genes were upregulated in tumors compared with adjacent normal tissue, and high tumor expression of Notch1 intercellular domain (NICD1) and the Notch target gene Hes1 correlated with poor chemotherapy response. Data mining of publicly available datasets confirmed that expression of Notch pathway genes is related to poor prognosis in osteosarcoma. On the basis of in vitro analysis, Notch signaling promoted osteosarcoma proliferation, enhanced chemoresistance, facilitated both migration and invasion, and upregulated stem cell-like characteristics. Xenograft models demonstrated that Notch signaling promotes primary tumor growth and pulmonary metastasis, and Notch inhibition is effective in reducing tumor size and preventing metastasis. Mechanistically, activated Notch signaling induces the expression of ephrinB1 and enhances the tumor-promoting ephrin reverse signaling. Overall, these findings provide functional evidence for Notch pathway genes as candidate biomarkers to predict prognosis in patients with osteosarcoma, and suggest a mechanistic rationale for the use of Notch inhibitors to treat osteosarcoma. IMPLICATIONS: The study provides preclinical evidence for Notch pathway as a molecular marker to predict osteosarcoma prognosis and as a therapeutic target against osteosarcoma. In addition, we identified a novel mechanism that ephrin reverse signaling acts as a key mediator of Notch pathway.

A Zingerone Analog, Acetyl Zingerone, Bolsters Matrisome Synthesis, Inhibits Matrix Metallopeptidases, and Represses IL-17A Target Gene Expression

Zingerone (Z) is a phenolic alkanone derived from natural sources with anti-inflammatory and antioxidant effects. Acetyl zingerone (AZ) is a recently designed molecule that shares structural features with Z but is expected to have improved stability and antioxidant function. This study utilized microarrays to compare the effects of Z and AZ on gene expression in reconstituted human epidermis. Both Z and AZ increased Notch pathway gene expression (NOTCH1 and MAML3) and decreased expression of genes linked to extracellular matrix disassembly (MMP3 and CTSV) and reactive oxygen species metabolism (PMAIP1 and ARG2). Although Z and AZ each inhibited invitro matrix metallopeptidase (MMP)-1, MMP-3, and MMP-12 activity, inhibition of MMP-3 and MMP-12 was greater with AZ. Moreover, AZ led to more consistent increases in the expression of genes encoding collagens (COL11A2), proteoglycans (VCAN), and extracellular matrix glycoproteins (SPARC). Finally, AZ opposed gene expression patterns associated with fibroblast senescence, keratinocyte differentiation, and IL-17A stimulation. These effects were AZ-specific and not replicated by Z. These results show that AZ improves extracellular matrix integrity with retinoid-like effects on differentiation and inflammation. Our findings provide a rationale for clinical studies to understand the benefits of AZ in the treatment or prevention of skin aging, or potentially, as a treatment for other human skin diseases.

Abstract 5164: Signaling crosstalk within prostate tumor microenvironment mediates castrate resistant disease progression

Abstract Juxtacrine signaling with the stromal compartment is important to the development of castrate resistance prostate cancer. We found that Notch signaling is upregulated in prostate cancer associated fibroblasts (CAFs) compared with normal tissue associated fibroblasts (NAFs). Further, inhibition of Notch signaling in CAFs resulted in decreased Wnt signaling and decrease in expression of Notch pathway genes. Interestingly, androgen receptor antagonism resulted in increased Notch signaling and the upregulation of downstream Wnts. Further, generation of chimeric tissue recombinant xenografts with human RV1 prostate cancer epithelia with mouse prostate stromal cells with activated Notch signaling that are derived from NICD transgenic mice resulted in increased tumor size compared with wild type control stromal recombinants. Conversely, recombinants derived from prostatic fibroblasts of RBPJ-knockout mice that have a loss of Notch signaling demonstrated diminished tumorigenesis, compared with xenografts with the wild type fibroblasts. In trying to determine the mediator of Wnt-associated paracrine prostate epithelial expansion, we found downstream YAP activation in the RV1 cells. In a reciprocal epithelial-to-fibroblast communication we found that the RV1 cells treated with conditioned media from NICD fibroblasts showed increased IL-6 expression. Conversely, conditioned media from prostate cancer cells caused increased Notch target gene expression in CAFs, which was blocked upon addition of IL-6 neutralizing antibody. Taken together our results suggest that there is a stromal-derived feed-back - feed-forward signaling loop involving Notch and Wnt ligands that activates YAP in the epithelia for paracrine IL-6-dependent Notch activity back in the stromal fibroblasts. Critically, disruption of this cross-talk in the fibroblasts can potentially limit the gain of stem features and castrate resistance of the adjacent epithelia in halting lethal progression of prostate cancer. Citation Format: Manisha Tripathi, Srinivas Nandana, Jen-Ming Huang, Manabu Kato, Rajeev Mishra, Leland Chung, Li Xin, Neil Bhowmick. Signaling crosstalk within prostate tumor microenvironment mediates castrate resistant disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5164.

Abstract 1291: Notch2 inhibition as a therapeutic intervention in osteosarcoma

Abstract Notch signaling pathway is a mediator of cell differentiation and is critical for normal bone development. Four functional Notch ligands DLL1 and DLL4, and JAG1 and JAG2 showed various levels of affinity for Notch1-4 receptors. Dose dependent Notch signaling activation or blocking is attributed to anchored or soluble form of Notch ligands binding. Interestingly, Notch pathway is shown to play a dual role, either oncogenic or tumor suppressive, depending on signal dose and tissue-context. To understand Notch signaling activities in osteosarcoma (OS), we first studied the cell surface levels of Notch receptor expression in OS cell lines and patient derived xenograft (pdx) models. Cytometry based screening of NOTCH1-4 receptors showed NOTCH2 as a predominant surface expressed protein in most of the samples. Interestingly, the canonical Notch target genes HES1 and DTX1 were higher (~5-fold) in OS cells compared to mesenchymal cells (MSC). Subsequently, we evaluated selected Notch pathway gene expression in 48 patients with recurrent/metastatic OS by RNAseq analysis. High expression of Notch pathway-related genes was seen in a subset of patients. NOTCH2 expression was comparable between primary and metastatic OS specimens, highest as compared to other Notch receptors and was significantly higher than normal tissue. Thus we aimed to elucidate soluble Notch ligands mediated blocking of Notch signaling using immunoglobulin-G bound DLL-1, -3, and -4 and JAG-1, and -2 in in vitro studies using OS cells. Soluble DLL1-Fc treatment increased OS cell death as compared to the other Notch ligands as well as control-Fc cells. Addition of soluble DLL1 resulted in the decreased expression of Notch downstream genes such as HES1, DTX1 and HEY1. Taken together, these findings identify soluble DLL1 as a potent Notch pathway inhibitor in OS in vitro and support further exploration of NOTCH2 as a potential therapeutic target in OS. Citation Format: Sankaranarayanan Kannan, John A. Livingston, Michael Roth, Jonathan Benjamin, Yifei Wang, Zhongting Zhang, Wendong Zhang, Chia-Chin Wu, Hannah Beird, Andrew Futreal, Richard Gorlick. Notch2 inhibition as a therapeutic intervention in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1291.

Tumour-Derived Laminin α5 (LAMA5) Promotes Colorectal Liver Metastasis Growth, Branching Angiogenesis and Notch Pathway Inhibition.

Hepatic metastatic growth is dependent upon stromal factors including the matrisomal proteins that make up the extracellular matrix (ECM). Laminins are ECM glycoproteins with several functions relevant to tumour progression including angiogenesis. We investigated whether metastatic colon cancer cells produce the laminins required for vascular basement membrane assembly as a mechanism for the promotion of angiogenesis and liver metastasis growth. qPCR was performed using human-specific primers to laminin chains on RNA from orthotopic human colorectal liver metastases. Laminin α5 (LAMA5) expression was inhibited in colon cancer cells using shRNA. Notch pathway gene expression was determined in endothelia from hepatic metastases. Orthotopic hepatic metastases expressed human laminin chains α5, β1 and γ1 (laminin 511), all of which are required for vascular basement membrane assembly. The expression of Laminin 511 was associated with reduced survival in several independent colorectal cancer cohorts and angiogenesis signatures or vessel density significantly correlated with LAMA5 expression. Colorectal cancer cells in culture made little LAMA5, but its levels were increased by culture in a medium conditioned by tumour-derived CD11b+ myeloid cells through TNFα/NFκB pathway signalling. Down-regulation of LAMA5 in cancer cells impaired liver metastatic growth and resulted in reduced intra-tumoural vessel branching and increased the expression of Notch pathway genes in metastasis-derived endothelia. This data demonstrates a mechanism whereby tumour inflammation induces LAMA5 expression in colorectal cancer cells. LAMA5 is required for the successful growth of hepatic metastases where it promotes branching angiogenesis and modulates Notch signalling.

Bacoside A Induced Sub-G0 Arrest and Early Apoptosis in Human Glioblastoma Cell Line U-87 MG through Notch Signaling Pathway.

BackgroundGlioblastoma multiforme (GBM) is a highly malignant brain tumor with a worst prognosis of less than one year despite advance treatment facilities. Among various signaling pathway genes displaying genetic modifications, aberrant expression of Notch pathway genes is frequent in GBM offering novel therapeutic targets. Herbal extracts having anticancer properties are used in adjuvant therapy that is safe and affordable as compared to chemotherapeutics. Bacopa monnieri has been used for the development of brain cells because of its neuroprotective properties. Its anticancer properties have shown to be promising in cancer treatment.MethodsThe anticancer properties of Bacoside A, an active and abundant component of Bacopa monnieri was assessed on U-87 MG cell line and its effects on expression of Notch pathway genes were studied. Cell cycle arrest and apoptosis were studied using flow cytometry. Expression of Notch pathway genes comprising of Notch receptors (notch1, notch2, notch3 and notch4), ligands (jagged1 and jagged2), a component of gamma-secretase complex (APH1A) and downstream target (HES1) were evaluated by quantitative real-time PCR.ResultsBacoside A exhibited considerable cytotoxicity on U-87 MG cells inducing cell cycle arrest and apoptosis. Cell cycle analysis revealed a significant arrest of 39.21% cells in sub-G0 phase at 80 µg/mL concentration, increasing to 53.21% at a higher concentration of 100 µg/mL. The fraction of early apoptotic cells in control was low (3.48%) that increased substantially to 31.36% and 41.11% after 80 µg/mL and 100 µg/mL of Bacoside A treatment respectively. Additionally, the expression of notch1 gene decreased after exposure to Bacoside A with a fold change of 0.05, whereas HES1 gene expression was increased by 25 fold.ConclusionThese data indicate that Bacoside A has a possible anticancer activity that could be inducing cell cycle arrest and apoptosis through Notch pathway in GBM in vitro.

Abstract A177: Role of promoter methylation in regulation of Notch4 and JAG2 gene expression in human glioblastoma

Abstract 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.

FOXP1 Promotes Embryonic Neural Stem Cell Differentiation by Repressing Jagged1 Expression

SummaryMutations in FOXP1 have been linked to neurodevelopmental disorders including intellectual disability and autism; however, the underlying molecular mechanisms remain ill-defined. Here, we demonstrate with RNA and chromatin immunoprecipitation sequencing that FOXP1 directly regulates genes controlling neurogenesis. We show that FOXP1 is expressed in embryonic neural stem cells (NSCs), and modulation of FOXP1 expression affects both neuron and astrocyte differentiation. Using a murine model of cortical development, FOXP1-knockdown in utero was found to reduce NSC differentiation and migration during corticogenesis. Furthermore, transplantation of FOXP1-knockdown NSCs in neonatal mice after hypoxia-ischemia challenge demonstrated that FOXP1 is also required for neuronal differentiation and functionality in vivo. FOXP1 was found to repress the expression of Notch pathway genes including the Notch-ligand Jagged1, resulting in inhibition of Notch signaling. Finally, blockade of Jagged1 in FOXP1-knockdown NSCs rescued neuronal differentiation in vitro. Together, these data support a role for FOXP1 in regulating embryonic NSC differentiation by modulating Notch signaling.

KrasG12D upregulates Notch signaling to induce gallbladder tumorigenesis in mice.

BackgroundKras mutations and increased Notch activation occur frequently in gallbladder cancer. However, their roles in gallbladder carcinogenesis have not been defined. This study was aimed at determining whether expression of mutant Kras was sufficient to induce gallbladder carcinoma and whether Notch deregulation played a role in this context.MethodsWe determined Cre recombination activity of Pdx1-Cre in the gallbladder using a reporter strain and examined gallbladder tumor development in the KrasLSL- G12D/+;Pdx1-Cre mice. We analyzed expression of Notch pathway genes in the mouse gallbladder by immunohistochemistry, quantitative RT-PCR, and Western blot analysis. We also determined the effect of Jag1 deletion on Kras-induced gallbladder tumor development.ResultsPdx1-Cre exhibits robust recombination activity in the gallbladder epithelium. KrasLSL-G12D/+;Pdx1-Cre mice form early onset adenoma in the gallbladder and adjacent biliary tract with complete penetrance, albeit short of invasive adenocarcinoma. KrasG12D upregulates expressions of Notch2, Notch3, Notch4, Jag1 and downstream target genes Hes1, Hey1 and Hey2, and deletion of Jag1 partially suppresses KrasG12D-induced adenoma development.ConclusionsKrasG12D induces gallbladder adenoma and Notch plays a key role in Kras-initiated gallbladder tumorigenesis.

Abstract 1332: NOTCH - HEY1 pathway induces EMT in head and neck squamous cell carcinoma

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is noted to have a NOTCH mutation rate of 20% within the Cancer Genome Atlas (TCGA) dataset consistent with tumor suppressor gene status. However, NOTCH genes are also upregulated in wild type HNSCC and show signs of downstream activation in a substantial proportion of HNSCC. In this study, we aimed to further explore the potential for NOTCH pathways to be activated in HNSCC. Material and Methods mRNA sequence of TCGA data includes 520 HNSCC tumors and 46 normal tissues. We used 447 HNSCC excluding 73 tumors with NOTCH mutations. The activated groups of NOTCH downstream genes were defined as tumors with expression 1 standard deviation above the mean of normal tissue. We used four HNSCC cell lines (SKN3, Cal27, SCC61, SCC090) to ascertain the results of the TCGA analysis in vitro. Result In TCGA analysis, HEY1 activated HNSCC had significant more expression of NOTCH pathway genes. In particular NOTCH4 had the most significant correlation with HEY1 activation among NOTCH receptors. To confirm this result in vitro, HEY1 expression was compared between si-control and si-NOTCH4 of SKN3, Cal27, SCC61 and SCC090. As expected, HEY1 expression in all si-NOTCH4 treated cell lines were significant decreased. Next, we examined the relationship about NOTCH4 and an epithelial mesenchymal transition phenotype (EMT). Using TCGA data, we found that mesenchymal marker genes were significantly increased in high NOTCH4 group. In vitro experiments, si-NOTCH4 cells decreased mesenchymal marker gene expression such as Fibronectin and TWIST1. We also examined HEY1 function. Similar to NOTCH4, it was found that HEY1 is significantly related to EMT in silico and in vitro. Conclusion The NOTCH4-HEY1 pathway is significantly upregulated in HNSCC and induces EMT. Citation Format: Takahito Fukusumi, Theresa W. Guo, Shuling Ren, Akihiro Sakai, Mizuo Ando, Sunny Haft, Chao Liu, Joseph A. Califano. NOTCH - HEY1 pathway induces EMT in head and neck squamous cell carcinoma [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 1332. doi:10.1158/1538-7445.AM2017-1332