Downregulation Of Notch Signaling Research Articles

Downregulating Notch Counteracts KrasG12D-Induced ERK Activation and Oxidative Phosphorylation in Myeloproliferative Neoplasm

Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions highly depend on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN).We used genetic approaches to down-regulate Notch signaling in KrasG12D/+ mice, which develop both T-ALL and MPN. Down-regulation of Notch signaling in hematopoietic cells is achieved through Mx1-Cre-mediated conditional expression of Rosa26-GFP-dnMAML1, which inhibits canonical Notch signaling, or conditional knockout of Pofut1, which catalyzes O-fucosylation of Notch receptors and modulates Notch receptor ligand interactions. To determine whether down-regulating Notch signaling prevents T-ALL and/or promotes MPN in a cell-autonomous manner, we transplanted the same number of KrasG12D/+, KrasG12D/+; Rosa26GFP-dnMAML1/+, or KrasG12D/+; Pofut1-/- bone marrow cells (CD45.2+) along with congeneic competitor cells (CD45.1+) into lethally irradiated mice (CD45.1+). As expected, inhibiting Notch signaling significantly blocked T-cell development and completely prevented T-ALL development in recipients; T-ALL that developed in a fraction of recipient mice were derived from rare donor cells that expressed oncogenic Kras and preserved intact Notch signaling. Surprisingly, we found that the percentage of donor-derived myeloid cells was significantly lower in recipients transplanted with KrasG12D/+; Rosa26GFP-dnMAML1/+ or KrasG12D/+; Pofut1-/- bone marrow cells and consequently none of them developed donor-derived MPN-like disease. In contrast, ~20% of the recipient mice transplanted with KrasG12D/+ cells developed a lethal, donor-derived MPN (P=0.02). Because the hematopoietic stem cell (HSC) frequency was significantly lower in KrasG12D/+; Rosa26GFP-dnMAML1/+ bone marrow than that in KrasG12D/+ bone marrow, we investigated whether the absence of MPN was due to the reduced HSC reconstitution in recipients. To normalize for HSC numbers, we transplanted lethally irradiated mice with same number of KrasG12D/+ or KrasG12D/+; Rosa26GFP-dnMAML1/+ splenocytes, which contained similar numbers of HSCs mobilized from the bone marrow. Consistent with our previous observation, only 1 out of 21 recipient mice with KrasG12D/+; Rosa26GFP-dnMAML1/+ cells developed a donor-derived (KrasG12D/+; Rosa26GFP-dnMAML1/+) MPN disease, while 6 out of 12 recipient mice with KrasG12D/+ cells died with donor-derived MPN (P=0.002). Of note, in 3 T-ALL free recipients with KrasG12D/+; Rosa26GFP-dnMAML1/+cells, although mutant cells were dominant in hematopoietic tissues, none of them displayed MPN phenotypes over the time. Together, our results indicate that blocking Notch signaling inhibits both T-ALL and MPN development in a cell-autonomous manner.Mechanistically, analysis of donor-derived hematopoiesis revealed that loss of Notch signaling significantly reduced myeloid compartment in Kras recipients with significant decrease of proliferation and cytokine-evoked ERK1/2 activation in Kras myeloid progenitor (MP) cells. RNA-Seq analysis of control and mutant MPs suggest that overexpression of DUSP1, a dual phosphatase that inactivates ERK, might lead to reduced ERK signaling in KrasG12D/+; Rosa26GFP-dnMAML1/+ MPs. Moreover, Kras MPs exhibited enhanced oxidative phosphorylation and mitochondria respiration, and this aberrant gene expression pattern was largely restored by DNMAML expression. These metabolic changes were further validated in functional assays. Our results demonstrate an essential role of Notch in oncogenic Kras-induced MPN through modulating ERK signaling and mitochondrial metabolism. DisclosuresNo relevant conflicts of interest to declare.

Notch Signaling in Bone Marrow Nestin-Expressing Cells Controls Balance of Erythropoiesis at the Bone Marrow and Spleen

[Background] Nestin-expressing cells (NeC) have been characterized as one of many types of bone marrow (BM) microenvironmental cells, including endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, etc. Recent studies have gradually provided information about anatomy and functions of each of these cells. Nevertheless, subcellular signaling and transcriptional regulations in individual miciroenvironmental cells have poorly been demonstrated. On a different line of studies, it has been suggested that NOTCH signaling in BM microenvironmental cells affects hematopoiesis; despite this, information is limited whether NOTCH signaling plays a role in NeC. It is of note that nestin was originally identified in neural stem cells (NSC), that NOTCH signaling is known to play a pivotal role in the NSC, and that the BM NeC could be derived from neuroectoderm. This potential linkage urged us to investigate whether and how downregulation of NOTCH signaling in BM NeC affects hematopoiesis.[Method] Mice with an rbpj-flox allele were crossed with those with a CreERT2/GFP (Green Fluorescent Protein) transgene under the nestin promoter. At 8-12 weeks, tamoxifen was intraperitoneally injected for 4-12 weeks to delete the rbpj gene only in NeC (rbpj cKO mice). In this experimental system, GFP is expected to be expressed as a surrogate marker for the rbpj gene deletion, by the deletion of stop codon inserted at 5' to the cDNA of GFP. Then, transplantation assays were performed using rbpj-null BM cells as a donor to reconstitute hematopoiesis in the wild-type mice, or using rbpj-null mice as recipients to see reconstitution of hematopoiesis from wild-type BM cells. The effect of splenectomy was investigated in the untransplanted and transplanted conditions. The littermate rbpjnull/wt orrbpjwt/wtmice were used as controls.[Results] GFP was detected in 0.1-0.5% of flow cytometry (FCM)-sorted CD45(-) cells only after tamoxifen injection. Deletion of rbpj was specifically confirmed in GFP-positive BM and spleen cells. Tamoxifen induced mild splenomegaly in rbpj cKO mice compared with littermate control mice. Enlarged spleen showed preserved follicular architecture but increased CD71(+)Ter119(+) mature erythroid cells in the red pulp. In contrast, BM of rbpj cKO mice bearing mild splenomegaly demonstrated marked decrease in the CD71(+)Ter119(+) mature erythroid cells without obvious anemia. There was a substantial animal-to-animal variation in the phenotypes; however, the strength of phenotypes was correlated with the frequency of GFP-positive cells in the BM, suggesting that the phenotypic variation was a result of the efficiency of rbpj gene deletion. We hypothesized that the rbpj cKO mice would develop anemia if spenectomized, because extramedullary erythropoiesis in spleen might compensate the defective BM erythropoiesis. However, tamoxifen did not cause significant anemia in splenectomized rbpj cKO mice. In these mice, reduction of the CD71(+)Ter119(+) mature erythroid cells in BM was significantly milder than nonsplenectomized mice. In transplantation analysis, the recipient rbpj cKO mice transplanted with BM cells from wild-type mice showed a reduction in CD71(+)Ter119(+) mature erythroid cells and mild splenomegaly, as were seen in rbpj cKO mice without transplantation. The phenotypes were again erased by the splenectomy. The recipient wild-type mice transplanted with BM cells from rbpjcKO mice did not show any phenotypes.[Discussion] Rbpj cKO in NeC induced impaired erythroid differentiation in BM together with mild splenomegaly. We confirmed that these phenotypes were caused by rbpj cKO in BM NeC by transplantation experiments. Surprisingly, such BM erythropoiesis impairment was reversed by splenectomy. This unexpected finding uncovered the presence of a previously unidentified balance controller between BM and spleen erythropoiesis. Hematopoietic stem cell-autonomous NOTCH signaling has been shown to be dispensable for adult murine hematopoiesis; however, NOTCH signaling in BM-NeC is responsible for control of balance of erythropoiesis at the BM and the spleen. DisclosuresObara:Alexion Pharmaceuticals: Honoraria, Research Funding.

Extensive Supporting Cell Proliferation and Mitotic Hair Cell Generation by In Vivo Genetic Reprogramming in the Neonatal Mouse Cochlea.

The generation of hair cells (HCs) from the differentiation of proliferating supporting cells (SCs) appears to be an ideal approach for replacing lost HCs in the cochlea and is promising for restoring hearing after damage to the organ of Corti. We show here that extensive proliferation of SCs followed by mitotic HC generation is achieved through a genetic reprogramming process involving the activation of β-catenin to upregulate Wnt signaling, the deletion of Notch1 to downregulate Notch signaling, and the overexpression of Atoh1 in Sox2(+) SCs in neonatal mouse cochleae. We used RNA sequencing to compare the transcripts of the cochleae from control mice and from mice with β-catenin activation, Notch1 deletion, and β-catenin activation combined with Notch1 deletion in Sox2(+) SCs. We identified the genes involved in the proliferation and transdifferentiation process that are either controlled by individual signaling pathways or by the combination of Wnt and Notch signaling. Moreover, the proliferation of SCs induced by Notch1 deletion disappears after deleting β-catenin in Notch1 knock-out Sox2(+) cells, which further demonstrates that Notch signaling is an upstream and negative regulator of Wnt signaling. We show here that the extensive proliferation of supporting cells (SCs) and the subsequent mitotic hair cell (HC) generation is achieved through a genetic reprogramming process involving activation of β-catenin to upregulate Wnt signaling, deletion of Notch1 to downregulate Notch signaling, and overexpression of Atoh1 in Sox2(+) SCs in neonatal mice cochleae. By comparing the transcripts of the cochleae among controls, β-catenin activation, Notch1 deletion, and β-catenin activation combined with Notch1 deletion group, we identified multiple genes involved in the proliferation and transdifferentiation process that are either controlled by individual signaling pathways or by the combination of Wnt and Notch signaling. This provides a better understanding of the mechanisms behind mitotic HC generation and might provide new approaches to stimulating mitotic HC regeneration.

Niclosamide inhibits colon cancer progression through downregulation of the Notch pathway and upregulation of the tumor suppressor miR-200 family.

Colorectal cancer (CRC) is among the most frequent causes of cancer-related deaths worldwide. Thus, there is a need for the development of new therapeutic approaches for the treatment of CRC. Accumulating evidence has revealed that niclosamide, an anthelminthic drug, exerts antitumor activity in several types of cancer, including colon cancer. However, the underlying molecular mechanisms responsible for the effects of this drug remain elusive. Previous studies have shown that the aberrant Notch signaling pathway contributes to the carcinogenesis of colon cancer. Herein, we examined the effects of niclosamide on the growth, migration and apoptosis of colon cancer cells, and the role of the Notch signaling pathway. By performing MTT, wound-healing and Transwell migration assays, we observed that niclosamide suppressed the growth and migration of colon cancer cells, and flow cytometry demonstrated that cell apoptosis was induced. This was associated with the decreased protein expression of Notch1, Notch2, Notch3 and Hey1, and the increased expression of the tumor suppressor microRNA (miR or miRNA)-200 family members (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) that are typically downregulated in colon cancer. Collectively, these findings demonstrate that niclosamide potentially inhibits the progression of colon cancer by downregulating Notch signaling and by upregulating the miR-200 family members.

Abstract 678: Lunatic Fringe and p53 cooperatively prevent development of claudin-low breast cancer

Abstract Claudin-low breast cancer (CLBC) is a poor prognosis disease that disproportionately affects younger patients and women with African ancestry, causing significant morbidity and mortality due to chemoresistance and lack of targeted therapy. CLBC shows stemness and mesenchymal features, and is thought to have originated from mammary stem cells. However, little is known on how these tumors initiate and progress. We previously reported that deletion of Lunatic Fringe (Lfng), a modulator of Notch signaling, in the moue mammary epithelial cells induced development of mammary tumors, among them two-thirds are basal-like breast cancer and one-third resemble human CLBC. Deletion of p53 in combination with other mutations (inactivation of Rb or overexpression of oncogenic c-Met) also resulted in mammary tumors with mesenchymal features. We hypothesize that Lfng and p53 may cooperate to suppress CLBC initiation and progression. To this end, we have generated mammary-specific deletion of Lfng and p53 using MMTV-Cre. The vast majority of Lfngflox/flox;p53flox/flox;MMTV-Cre mice succumbed to lymphoma by 3-5 months of age before mammary tumor onset, likely due to the leaky deletion in B- and T cells. Interestingly, deletion of both copies of Lfng and one copy of p53 (Lfngflox/flox;p53flox/+;MMTV-Cre) dramatically accelerated mammary tumor development compared to the Lfngflox/flox;MMTV-Cre model, with the median tumor-free survival shortened from 600 days to 191 days. Of note, all mammary tumors from the Lfngflox/flox;p53flox/+;MMTV-Cre mice were consisted of mesenchymal/spindle-shaped cells and expressed markers of epithelial-mesenchymal transition (EMT), suggesting that deletion of one copy of p53 caused a shift of tumor subtype from basal-like to claudin-low. We found that deletion of Lfng caused up-regulation of E-cadherin in the mammary gland, whereas additional deletion of one copy of p53 decreased E-cadherin but dramatically increased vimentin levels, suggesting that p53 haploinsufficiency promotes EMT in this context. Compared with the wild type gland at 3 months of age, Lfngflox/flox;p53flox/+;MMTV-Cre mutant gland showed down-regulation of Notch downstream targets including Hes5, Hey1, and Hey2, suggesting reduced Notch signaling. Given that Notch signaling is known to restrict mammary stem cell expansion, down-regulation of Notch signaling may lead to inappropriate accumulation of mammary stem cells during CLBC initiation. Taken together, our results reveal genetic interaction between Notch and p53 in the pathogenesis of CLBC. Moreover, we have established a new mouse model for CLBC with complete penetrance and accelerated tumor onset, providing an ideal tool in defining molecular events leading to the initiation and progression of CLBC as well as identifying potential therapeutic targets for this poor prognosis disease. Citation Format: Wen-Cheng Chung, Shubing Zhang, Keli Xu. Lunatic Fringe and p53 cooperatively prevent development of claudin-low breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 678.

4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation.

Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress-mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress-activated Notch signaling to modulate trabeculation.

4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation.

Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress-mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress-activated Notch signaling to modulate trabeculation.

The Dmp1‐SOST Transgene Interacts With and Downregulates the Dmp1‐Cre Transgene and the RosaNotch Allele

Activation of Notch1 in osteocytes of Rosa(Notch) mice, where a loxP-flanked STOP cassette and the Nicd coding sequence were targeted to the reverse orientation splice acceptor (Rosa)26 locus, causes osteopetrosis associated with suppressed Sost expression and enhanced Wnt signaling. To determine whether Sost downregulation mediates the effects of Notch activation in osteocytes, Rosa(Notch) mice were crossed with transgenics expressing Cre recombinase or SOST under the control of the dentin matrix protein (Dmp)1 promoter. Dmp1-SOST transgenics displayed vertebral osteopenia and a modest femoral cancellous and cortical bone phenotype, whereas hemizygous Dmp1-Cre transgenics heterozygous for the Rosa(Notch) allele (Dmp1-Cre;Rosa(Notch)) exhibited osteopetrosis. The phenotype of Notch activation in osteocytes was prevented in Dmp1-Cre;Rosa(Notch) mice hemizygous for the Dmp1-SOST transgene. The effect was associated with downregulated Notch signaling and suppressed Dmp1 and Rosa26 expression. To test whether SOST regulates Notch expression in osteocytes, cortical bone cultures from Dmp1-Cre;Rosa(Notch) mice or from Rosa(Notch) control littermates were exposed to recombinant human SOST. The addition of SOST had only modest effects on Notch target gene mRNA levels and suppressed Dmp1, but not Cre or Rosa26, expression. These findings suggest that prevention of the Dmp1-Cre;Rosa(Notch) skeletal phenotype by Dmp1-SOST is not secondary to SOST expression but to interactions among the Dmp1-SOST and Dmp1-Cre transgenes and the Rosa26 locus. In conclusion, the Dmp1-SOST transgene suppresses the expression of the Dmp1-Cre transgene and of Rosa26.

Ginsenoside Rb1 inhibits matrix metalloproteinase 13 through down-regulating Notch signaling pathway in osteoarthritis.

Mounting evidence suggests that an excess of matrix metalloproteinase-13 (MMP-13) plays an important role in the breakdown of extracellular matrix in osteoarthritis (OA). Here, the effects of ginsenoside Rb1 (GRb1) on the expression of MMP-13 in IL-1β-induced SW 1353 chondrosarcoma cells and an experimental rat model of OA induced by anterior cruciate ligament transection (ACLT) were investigated. SW1353 chondrosarcoma cells were pretreated with or without GRb1 and Notch signaling pathway inhibitor, DAPT, then were stimulated with IL-1β. In rats, experimental OA was induced by ACLT. These rats then received intra-articular injections of vehicle, an inhibitor of γ-secretase, DAPT, and/or GRb1. Expression of MMP-13, collagen type II (CII), Notch1, and jagged 1 (JAG1) were verified by western blotting and immunohistochemistry. In addition, levels of MMP-13 mRNA were detected using quantitative real-time PCR. In histological analyses, treatment with DAPT reduced the number of cartilage lesions present and the expressions of MMP-13, CII, Notch1, and JAG1. In addition, treatment with GRb1 was associated with lower levels of Notch1 and JAG1 in both IL-1β-induced SW1353 chondrosarcoma cells and in the rat OA model. Furthermore, the suppressive effect of GRb1 on MMP-13 was greater than that exhibited by the signaling pathway inhibitor. In conclusion, GRb1 inhibits MMP-13 through down-regulating Notch signaling pathway in OA.

Gene expression profile analyze the molecular mechanism of CXCR7 regulating papillary thyroid carcinoma growth and metastasis.

BackgroundTo detect genetic expression profile alterations after papillary thyroid carcinoma (PTC) cells transfected with chemokine receptor CXCR7 gene by gene microarray, and gain insights into molecular mechanisms of how CXCR7 regulating PTC growth and metastasis.MethodsThe Human OneArray microarray was used for a complete genome-wide transcript profiling of CXCR7 transfected PTCs (K1-CXCR7 cells), defined as experimental group. Non CXCR7 transfected PTCs (K1 cells) were used as control group. Differential analysis for per gene was performed with a random variance model and t test, p values were adjusted to control the false discovery rate. Gene ontology (GO) on differentially expressed genes to identify the biological processes in modulating the progression of papillary thyroid carcinoma. Pathway analysis was used to evaluate the signaling pathway that differentially expressed genes were involved in. In addition, quantitative real-time polymerase chain reaction (q-PCR) and Western blot were used to verify the top differentially expression genes.ResultsComparative analysis revealed that the expression level of 1149 genes was changed in response to CXCR7 transfection. After unsupervised hierarchical clustering analysis, 270 differentially expressed genes were filtered, of them 156 genes were up-regulated whereas 114 genes were down-regulated in K1-CXCR7 cells. GO enrichment analysis revealed the differentially expressed genes were mainly involved in biopolymer metabolic process, signal transduction and protein metabolism. Pathway enrichment analysis revealed differentially expressed genes were mainly involved in ECM-receptor interaction, Focal adhesion, MAPK signaling pathway and Cytokine-cytokine receptor interaction pathway. More importantly, the expression level of genes closely associated with tumor growth and metastasis was altered significantly in K1-CXCR7 cells, including up-regulated genes FN1, COL1A1, COL4A1, PDGFRB, LTB, CXCL12, MMP-11, MT1-MMP and down-regulated genes ITGA7, and Notch-1.ConclusionsGene expression profiling analysis of papillary thyroid carcinoma can further delineate the mechanistic insights on how CXCR7 regulating papillary thyroid carcinoma growth and metastasis. CXCR7 may regulate growth and metastasis of papillary thyroid carcinoma via the activation of PI3K/AKT pathway and its downstream NF-κB signaling, as well as the down-regulation of Notch signaling.

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.

Downregulating Notch Signaling in KrasG12D/+ Mice Inhibits Both T-Cell Leukemia and Myeloproliferative Neoplasm in a Cell-Autonomous Manner

Notch signaling is implicated in diverse functions in hematopoiesis, including stem cell maintenance, cell fate specification, cell proliferation, and apoptosis. Aberrant Notch signaling is associated with the pathogenesis of various hematopoietic malignancies. NOTCH1 mutations that lead to elevated intracellular Notch1 activity are identified in 50-60% of patients with acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) and in 100% of oncogenic Ras-induced T-ALL in mice. Although Notch1 mutations contribute to the malignant transformation of normal T-cells to T-cell leukemia/lymphoma initiating cells in the KrasG12D/+-induced T-ALL model, it remains unclear whether Notch1 signaling is necessary for T-ALL genesis. Moreover, it remains controversial whether loss of Notch signaling promotes myeloproliferative neoplasms (MPN) in a cell-autonomous manner.To address these questions, we used genetic approaches to downregulate Notch signaling in KrasG12D/+ mice, which develop both T-ALL and MPN. Downregulation of Notch signaling in hematopoietic cells is achieved through Mx1-Cre–mediated conditional expression of Rosa26-GFP-dnMAML1, which inhibits canonical Notch signaling, or conditional knockout of Pofut1, which catalyzes O-fucosylation of Notch receptors and modulates Notch receptor ligand interactions. We found that either overexpression of dnMAML1 or deletion of Pofut1 significantly enhanced MPN phenotypes and shortened the survival of KrasG12D/+ mice. However, several pieces of evidence suggest that downregulation of Notch signaling in non-hematopoietic cells might influence MPN development. First, 100% of compound mice developed atopic dermatitis-like disease that is shown to promote MPN in a cell non-autonomous manner. Second, ~30% of endothelial cells were GFP-positive (expressing dnMAML1) in KrasG12D/+; Rosa26GFP-dnMAML1/+ mice.To determine whether downregulating Notch signaling prevents T-ALL and/or promotes MPN in a cell-autonomous manner, we transplanted the same number of KrasG12D/+, KrasG12D/+; Rosa26GFP-dnMAML1/+, or KrasG12D/+; Pofut1-/- bone marrow cells (CD45.2+) along with congeneic competitor cells (CD45.1+) into lethally irradiated mice (CD45.1+). As expected, inhibiting Notch signaling significantly blocked T-cell development and completely prevented T-ALL development in recipients; T-ALL that developed in a fraction of recipient mice were derived from rare donor cells that expressed oncogenic Kras and preserved intact Notch signaling. Surprisingly, we found that the percentage of donor-derived myeloid cells was significantly lower in recipients transplanted with KrasG12D/+; Rosa26GFP-dnMAML1/+ or KrasG12D/+; Pofut1-/- bone marrow cells and consequently none of them developed donor-derived MPN-like disease. In contrast, ~20% of the recipient mice transplanted with KrasG12D/+ cells developed a lethal, donor-derived MPN (P=0.02). Because the hematopoietic stem cell (HSC) frequency was significantly lower in KrasG12D/+; Rosa26GFP-dnMAML1/+ bone marrow than that in KrasG12D/+ bone marrow, we investigated whether the absence of the MPN was due to the reduced HSC reconstitution in recipients. To normalize for HSC numbers, we transplanted lethally irradiated mice with same number of KrasG12D/+ or KrasG12D/+; Rosa26GFP-dnMAML1/+ splenocytes, which contained similar numbers of HSCs mobilized from the bone marrow. Consistent with our previous observation, only 1 out of 12 recipient mice with KrasG12D/+; Rosa26GFP-dnMAML1/+ cells developed a donor-derived (KrasG12D/+; Rosa26GFP-dnMAML1/+) MPN disease, while 6 out of 12 recipient mice with KrasG12D/+ cells died with donor-derived MPN (P=0.02). Together, our results indicate that blocking Notch signaling inhibits both T-ALL and MPN development in a cell-autonomous manner. We are currently working on the underlying mechnisms. DisclosuresNo relevant conflicts of interest to declare.

Notch signaling regulates col1α1 and col1α2 expression in airway fibroblasts.

Subepithelial fibrosis is one of the common pathological features of asthmatic airway remodeling. During subepithelial fibrosis, type I collagen becomes the most abundant extracellular protein component. Studies have shown that Notch signaling participates in the progression of fibrosis; however, whether Notch signaling is involved in regulating type I collagen expression in airway fibroblasts remains unclear. The aim of the present study was to examine whether Notch signaling can regulate type I collagen expression in airway fibroblasts and to explore the underlying molecular mechanisms. Here, the expression of Notch signaling components was examined in mouse L929 cells and human MRC-5 cells. After upregulating or downregulating Notch signaling in these cell lines, col1α1 and col1α2 expression was examined. Using gene reporter assays, site-directed mutagenesis, and ChIP assays, the role of Hes1 binding sites in both the mouse and human COL1A1 and COL1A2 promoters was investigated. This study revealed that Notch signaling-related molecules (including Notch1, Hes1, and others) are expressed in L929 and MRC-5 cells and that Notch signaling regulates the expression of col1α1 and col1α2 in both cell lines. Additionally, over-expression of the Notch intracellular domain resulted in activation of the COL1A1 and COL1A2 promoters, and site-directed mutagenesis reporter assays revealed that Hes1 proteins might augment both mouse and human COL1A1 and COL1A2 promoter activity. Furthermore, ChIP assays confirmed that Hes1 binds to the COL1A1 and COL1A2 promoters in both L929 and MRC-5 cells. Therefore, it is reasonable to assume that Notch signaling can directly upregulate COL1A1 and COL1A2 promoter activity through a Hes1-dependent mechanism, which could serve as a possible target for pharmacotherapy of airway subepithelial fibrosis.

Cadherin-based adhesions in the apical endfoot are required for active Notch signaling to control neurogenesis in vertebrates

The development of the vertebrate brain requires an exquisite balance between proliferation and differentiation of neural progenitors. Notch signaling plays a pivotal role in regulating this balance, yet the interaction between signaling and receiving cells remains poorly understood. We have found that numerous nascent neurons and/or intermediate neurogenic progenitors expressing the ligand of Notch retain apical endfeet transiently at the ventricular lumen that form adherens junctions (AJs) with the endfeet of progenitors. Forced detachment of the apical endfeet of those differentiating cells by disrupting AJs resulted in precocious neurogenesis that was preceded by the downregulation of Notch signaling. Both Notch1 and its ligand Dll1 are distributed around AJs in the apical endfeet, and these proteins physically interact with ZO-1, a constituent of the AJ. Furthermore, live imaging of a fluorescently tagged Notch1 demonstrated its trafficking from the apical endfoot to the nucleus upon cleavage. Our results identified the apical endfoot as the central site of active Notch signaling to securely prohibit inappropriate differentiation of neural progenitors.

GDE2 downregulates Notch signaling to promote neurogenesis through GPI‐anchor cleavage (223.1)

The six‐transmembrane protein GDE2 induces the differentiation of postmitotic motor neurons by downregulating Notch in adjacent motor neuron progenitors. GDE2 function depends upon the integrity of its extracellular enzymatic domain that is homologous to bacterial glycerophosphodiester phosphodiesterases (GDPD). GDPDs metabolize glycerophosphodiesters into glycerol‐3‐phosphate and their corresponding alcohols, but it is unclear if and how GDE2 utilizes this mechanism to inhibit Notch activation. In this talk, I will discuss our recent observations that GDE2 regulates Notch signaling non cell autonomously, and that it does so through novel enzymatic mechanisms that control the surface availability of the Notch ligand Dll1.

FGF and Notch signaling hierarchy in sensory neurogenesis (651.18)

The purpose of this study was to determine the regulatory relationship of sensory neurogenesis in the trigeminal placode between the Fibroblast Growth Factor (FGF) and Notch signaling pathways. To do this, FGF signaling was blocked cell autonomously or globally while simultaneously downregulating Notch signaling in 10‐12 somite stage Gallus gallus chick embryos. Cell autonomous FGF inhibition with global Notch downregulation significantly reduced the number of proneural cells contributing the future trigeminal ganglion (p=0.0002). A similar reduction was found with concurrent global inhibition of FGF and Notch (p<0.0001). Remarkably, blocking FGF either cell autonomously or globally with simultaneous Notch downregulation reversed the effects of robust neurogenesis seen with Notch downregulation alone (p<0.0001). Further, Notch downregulation for 4 or 12 hours showed no difference in the level of FGF‐receptor 4 mRNA expression when compared to controls. It did however result in a highly fragmented basal lamina, which was reversed when blocking FGF signaling. From these data, we invalidate the FGF downregulates Notch in sensory neurogenesis model and propose a mechanism for parallel regulation of FGF and Notch leading to basement membrane fragmentation and subsequent delamination of differentiating opV placode cells.Grant Funding Source: Supported by NIH/NICHD ‐ R01 HD046475

Distinct Notch signaling outputs pattern the developing arterial system

Differentiation of arteries and veins is essential for the development of a functional circulatory system. In vertebrate embryos, genetic manipulation of Notch signaling has demonstrated the importance of this pathway in driving artery endothelial cell differentiation. However, when and where Notch activation occurs to affect endothelial cell fate is less clear. Using transgenic zebrafish bearing a Notch-responsive reporter, we demonstrate that Notch is activated in endothelial progenitors during vasculogenesis prior to blood vessel morphogenesis and is maintained in arterial endothelial cells throughout larval stages. Furthermore, we find that endothelial progenitors in which Notch is activated are committed to a dorsal aorta fate. Interestingly, some arterial endothelial cells subsequently downregulate Notch signaling and then contribute to veins during vascular remodeling. Lineage analysis, together with perturbation of both Notch receptor and ligand function, further suggests several distinct developmental windows in which Notch signaling acts to promote artery commitment and maintenance. Together, these findings demonstrate that Notch acts in distinct contexts to initiate and maintain artery identity during embryogenesis.

Pretreatment with the γ-secretase inhibitor DAPT sensitizes drug-resistant ovarian cancer cells to cisplatin by downregulation of Notch signaling

Notch signaling is implicated in ovarian cancer tumorigenesis and inhibition of Notch signaling with γ-secretase inhibitor DAPT resulted in reduction of tumor cell viability and induction of apoptosis in ovarian cancer cells. This study investigated whether DAPT has the same effect on ovarian cancer cells that are resistant to cisplatin and the underlying molecular events. Ovarian cancer cell lines resistant to cisplatin were treated with DAPT, cisplatin or combination for cell viability MTT, flow cytometric cell cycle, ELISA apoptosis and colony formation assays. qRT-PCR and western blotting were used to detect gene expressions. We found that pretreatment of ovarian cancer cisplatin-resistant cell lines with DAPT for 24 h and then with cisplatin for 72 h showed a synergistic antitumor activity in these cell lines, while cisplatin treatment and then addition of DAPT just showed an additive or antagonistic effects on these cisplatin-resistant ovarian cancer cells. Moreover, pretreatment of ovarian cancer cell lines with DAPT and then with cisplatin also inhibited tumor cell colony formation capacity, arrested tumor cells at G2 phase of the cell cycle and induced apoptosis. The cell cycle and apoptosis-related genes, such as cyclin B1, Bcl-2 and caspase-3, were also modulated by the treatment. Pretreatment of ovarian cancer cell lines with DAPT and then with cisplatin downregulated Notch1 and Hes1 expression dose- and time-dependently. The current data demonstrate that DAPT pretreatment was able to sensitize cisplatin-resistant human ovarian cancer cells to cisplatin by downregulation of Notch signaling.

Effects of Notch Signaling on Regulation of Myeloid Cell Differentiation in Cancer

Functionally altered myeloid cells play an important role in immune suppression in cancer, in angiogenesis, and in tumor cells' invasion and metastases. Here, we report that inhibition of Notch signaling in hematopoietic progenitor cells (HPC), myeloid-derived suppressor cells (MDSC), and dendritic cells is directly involved in abnormal myeloid cell differentiation in cancer. Inhibition of Notch signaling was caused by the disruption of the interaction between Notch receptor and transcriptional repressor CSL, which is normally required for efficient transcription of target genes. This disruption was the result of serine phosphorylation of Notch. We demonstrated that increased activity of casein kinase 2 (CK2) observed in HPC and in MDSC could be responsible for the phosphorylation of Notch and downregulation of Notch signaling. Inhibition of CK2 by siRNA or by pharmacological inhibitor restored Notch signaling in myeloid cells and substantially improved their differentiation, both in vitro and in vivo. This study demonstrates a novel mechanism regulation of Notch signaling in cancer. This may suggest a new perspective for pharmacological regulation of differentiation of myeloid cells in cancer.

Pterostilbene Exerts Antitumor Activity via the Notch1 Signaling Pathway in Human Lung Adenocarcinoma Cells

Although pterostilbene (PTE) has been shown to have potent antitumor activities against various cancer types, the molecular mechanisms of these activities remain unclear. In this study, we investigated the antitumor activity of PTE against human lung adenocarcinoma in vitro and in vivo and explored the role of the Notch1 signaling pathway in this process. PTE treatment resulted in a dose- and time-dependent decrease in the viability of A549 cells. Additionally, PTE exhibited strong antitumor activity, as evidenced not only by a reduced mitochondrial membrane potential (MMP) and a decreased intracellular glutathione content but also by increases in the apoptotic index and the level of reactive oxygen species (ROS). Furthermore, PTE treatment induced the activation of the Notch1 Intracellular Domain (NICD) protein and activated Hes1. DAPT (a gamma secretase inhibitor) and Notch1 siRNA prevented the induction of NICD and Hes1 activation by PTE treatment and sensitized the cells to PTE treatment. The down-regulation of Notch signaling also prevented the activation of pro-survival pathways (most notably the PI3K/Akt pathway) after PTE treatment. In summary, lung adenocarcinoma cells may enhance Notch1 activation as a protective mechanism in response to PTE treatment. Combining a gamma secretase inhibitor with PTE treatment may represent a novel approach for treating lung adenocarcinoma by inhibiting the survival pathways of cancer cells.