Notch Induction Research Articles

Increased AXLhigh myeloid cells as pathognomonic marker in Langerhans cell histiocytosis and Langerin expression dependence of mTOR inhibition

Langerhans cell histiocytosis (LCH) is characterized by an expansion and accumulation of pathological histiocytes expressing langerin (CD207) and CD1a in different organs under an inflammatory milieu. The origin of pathognomonic precursors of LCH is widely debated, but monocytes and pre-dendritic cells (pre-DC) play a significant role. Remarkably, we found an expansion of AXLhigh cells in the CD11c+ subset of patients with active LCH, which also express the pathognomonic CD207 and CD1a. Moreover, we obtained a monocyte-derived LC-like (mo-LC-like) expressing high levels of AXL when treated with inflammatory cytokine, or plasma of patients with active disease. Intriguingly, inhibiting the mTOR pathway at the initial stages of monocyte differentiation to LC-like fosters the pathognomonic LCH program, highly increasing CD207 levels, together with NOTCH1 induction. We define here that AXLhigh could also be taken as a strong pathognomonic marker for LCH, and the release of Langerin and NOTCH1 expression depends on the inhibition of the mTOR pathway.

Selected Articles from This Issue

Neratinib is an irreversible pan-HER inhibitor approved for patients with HER2 amplified breast cancer. MutHER trial evaluated the antitumor activity of neratinib and neratinib in combination with fulvestrant for HER2 non-amplified metastatic breast cancer harboring activating mutations in HER2, a population otherwise not eligible for HER2-targeted therapy. The study demonstrated the antitumor effect of neratinib, especially in invasive lobular breast cancer. Interestingly, responses were observed with the addition of trastuzumab to neratinib upon disease progression, suggesting dual HER2 blockade may be important in targeting HER2 mutation. Further investigation of trastuzumab in combination with neratinib is therefore warranted.Fusions involving the NTRK1/2/3 genes have been identified in many tumor types, several of which often metastasize to the CNS. Entrectinib is a potent TRK inhibitor that was specifically designed to penetrate and remain in the CNS. Demetri and colleagues present the latest results of an integrated analysis of three phase I/II trials: entrectinib continued to induce deep and durable systemic and intracranial responses across tumor types and was well tolerated. Entrectinib could address the need for a CNS-active treatment in NTRK fusion-positive solid tumors; therefore, encouraging broader NTRK fusion testing would improve patient access to effective, personalized treatments.Concurrent radiation therapy (RT) with cetuximab, an anti-EGFR monoclonal antibody (mAb), is a standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, control in high-risk disease is suboptimal. CTLA-4+ T regulatory cells (Treg) dampen cellular immunity and correlate negatively with clinical outcomes. Ferris and colleagues conducted a phase I trial to evaluate the safety and preliminary efficacy of the CTLA-4 targeting mAb, ipilimumab, with standard, fixed cetuximab-RT. They identified the recommended phase II dose and observed acceptable PFS and OS without cytotoxic chemotherapy. High expression of coinhibitory receptors PD1/LAG3/CD39 on baseline tumor infiltrating Treg was marginally associated with worse PFS. This is the first trial to combine CTLA-4 targeting with definitive cetuximab-RT in HNSCC, integrating immune checkpoint inhibitors into curative-intent RT platforms.Otani and colleagues used immunofluorescence staining and RNA-seq to uncover a significant induction of MDSC cell populations into brains of tumor-bearing mice treated with oHSV. This was mediated by NOTCH induction. In human patients, NOTCH signaling also correlated with a higher myeloid cell infiltration. The tumor infiltrating myeloid cells further spread NOTCH signaling in tumors and also created a CCL-2 feed-forward loop to induce more myeloid cells into the tumors. Increased CCL2 was also observed in serum of GBM patients treated with oHSV. Blockade of NOTCH signaling rescued these immunosuppressive effects and enhanced antitumor immunity.

Effect of the Notch1-mediated PI3K-Akt-mTOR pathway in human osteosarcoma.

Purpose: Osteosarcoma is one of the most common malignant bone tumours in early adolescence. The incidence rate of osteosarcoma has stagnated over the past 30 years, highlighting the need to develop novel therapies. In osteosarcoma cells, Notch1 expression is absent, and the Notch1 pathway is related to cancer cell proliferation, apoptosis and autophagy. Our study aimed to investigate the role of Notch1 in osteosarcoma development.Methods: We measured NICD1 expression induced by doxycycline treatment at various concentrations. The viability of human osteosarcoma cells (MG-63) induced by doxycycline was measured. Flow cytometry and cell apoptosis analysis were conducted to measure the effect of Notch1 on the cell cycle of human osteosarcoma cells. We also used a GFP-LC3 plasmid to detect Notch1-induced autophagy in MG-63 cells. Western blotting was conducted to analyse expression of the PI3K/Akt/mTOR signalling pathway through Notch1 induction by doxycycline.Results: In this study, we demonstrated that Notch1 activation by doxycycline potently suppressed cell proliferation by inducing S phase arrest in osteosarcoma cells. Doxycycline-induced Notch1 activation also induced apoptosis and autophagy in osteosarcoma cells. Moreover, we found that Notch1 inhibited PI3K/Akt/mTOR signalling to induce apoptosis and autophagy.Conclusion: In summary, our results revealed that Notch1 activation by doxycycline induces S phase arrest, apoptosis and autophagy by blocking PI3K/Akt/mTOR signalling in human osteosarcoma cells. Notch1 may be a potential clinical antitumour target for osteosarcoma therapy.

Context-Specific Osteogenic Potential of Mesenchymal Stem Cells.

Despite the great progress in the field of bone tissue regeneration, the early initiating mechanisms of osteogenic differentiation are not well understood. Cells capable of osteogenic transformation vary from mesenchymal stem cells of various origins to mural cells of vessels. The mechanisms of pathological calcification are thought to be similar to those of bone formation. Notch signaling has been shown to play an important role in osteogenic differentiation, as well as in pathological calcification. Nevertheless, despite its known tissue- and context-specificity, the information about its role in the osteogenic differentiation of different cells is still limited. We compared mesenchymal stem cells from adipogenic tissue (MSCs) and interstitial cells from the aortic valve (VICs) by their ability to undergo Notch-dependent osteogenic differentiation. We showed differences between the two types of cells in their ability to activate the expression of proosteogenic genes RUNX2, BMP2, BMP4, DLX2, BGLAP, SPRY, IBSP, and SPP1 in response to Notch activation. Untargeted metabolomic profiling also confirms differences between MSCs and VICs in their osteogenic state. Analysis of the activity of RUNX2 and SPP1 promoters shows fine-tuned dose-dependency in response to Notch induction and suggests a direct link between the level of Notch activation, and the proostogenic gene expression and corresponding osteogenic induction. Our data suggest that osteogenic differentiation is a context-dependent process and the outcome of it could be cell-type dependent.

MUC1-C Activates the BAF (mSWI/SNF) Complex in Prostate Cancer Stem Cells.

The Brg/Brahma-associated factor (BAF, mSWI/SNF) chromatin remodeling complex is of importance in development and has been linked to prostate oncogenesis. The oncogenic MUC1-C protein promotes lineage plasticity in the progression of neuroendocrine prostate cancer (NEPC), however, there is no known association between MUC1-C and BAF. We report here that MUC1-C binds directly to the E2F1 transcription factor and that the MUC1-C→E2F1 pathway induces expression of embryonic stem cell-specific BAF (esBAF) components BRG1, ARID1A, BAF60a, BAF155, and BAF170 in castrate-resistant prostate cancer (CRPC) and NEPC cells. In concert with this previously unrecognized pathway, MUC1 was associated with increased expression of E2F1 and esBAF components in NEPC tumors as compared with CRPC, supporting involvement of MUC1-C in activating the E2F1→esBAF pathway with progression to NEPC. MUC1-C formed a nuclear complex with BAF and activated cancer stem cell (CSC) gene signatures and the core pluripotency factor gene network. The MUC1-C→E2F1→BAF pathway was necessary for induction of both the NOTCH1 effector of CSC function and the NANOG pluripotency factor, and collectively, this network drove CSC self-renewal. These findings indicate that MUC1-C promotes NEPC progression by integrating activation of E2F1 and esBAF with induction of NOTCH1, NANOG, and stemness. SIGNIFICANCE: These findings show that MUC1-C, which promotes prostate cancer progression, activates a novel pathway that drives the BAF remodeling complex, induces NOTCH1 and NANOG, and promotes self-renewal of prostate cancer stem cells.

TAMI-11. IMPACT OF oHSV ACTIVATED NOTCH SIGNALING IN TUMOR MICROENVIRONMENT, AND ITS IMPACT ON ANTI-TUMOR IMMUNITY

Abstract NOTCH signaling is a method of cell-cell communication where membrane bound NOTCH ligands on signal-sending cells can bind to and initiate cleavage of the NOTCH receptor, releasing NICD which can initiate signal transduction in adjacent “signal-receiving” cells. We have recently shown that oHSV treatment of GBM cells induces NICD cleavage and NOTCH activation in adjacent uninfected glioma cells. RNA sequencing of GBM cells post-infection also uncovered Gene Ontology NOTCH signaling pathway to be significantly upregulated. This activation was induced by viral miRNA-H16, which represses FIH-1 expression. FIH-1 was found to be a negative regulator of Mib1, a ubiquitin ligase, which activates NOTCH ligand-mediated activation of adjacent signal-receiving cells bearing the NOTCH receptor (Otani et al Clin. Can. Res. 2020). Here we have investigated the impact of oHSV-induced NOTCH signaling on the tumor microenvironment. Treatment of brain tumors in immune competent mice with oHSV and NOTCH blocking gamma secretase inhibitor (GSI) induced an anti-tumor memory immune response. Long term survivors in mice treated with the combination also completely rejected subsequent tumor re-challenge in the other hemisphere. UMAP of flow cytometry of tumor-bearing hemispheres and functional analysis of isolated cellular fractions from treated mice showed a significant influx of MDSC cells after oHSV treatment that was rescued in mice treated with oHSV and GSI. Ongoing mechanistic studies are uncovering a significant induction of NOTCH in tumor associated macrophages that aids in recruitment of MDSC cells. Overall these studies have uncovered a significant impact of oHSV therapy on GBM tumor microenvironment and presents opportunities for combination therapies that can help improve therapeutic benefit and anti-tumor immunity.

TMEM45A is involved in renal fibrosis in rats by regulating Jagged1/Notch pathway.

Fibrosis, or the excess deposition of fibrous tissue, is a critical feature of chronic kidney disease. Here, using renal fibrotic rat as a model, which was established via 5/6 nephrectomy (Nx), the role of TMEM45A transmembrane protein in renal fibrosis was investigated. The results indicated that 5/6 Nx gradually led to histopathological abnormalities and loss of kidney function in rats, which correlated with upregulation of TMEM45A and Notch1. Interestingly, in NRK-49F renal cells, overexpression of TMEM45A resulted in up-regulation of extracellular matrix (ECM) components as well as induction of Notch-1 and Jagged-1. These effects were weakened by DAPT, an inhibitor of the Notch pathway, suggesting an important role of Notch signaling in mediating the functions of TMEM45A in NRK-49F cells Moreover, TMEM45A knockdown by TMEM45A siRNA in NRK-49F cells diminished TGF-b1-induced upregulation of ECM components, inflammatory cytokines, Notch-1 and Jagged-1. Correspondingly, TGF-beta 1 exhibited pro-fibrogenic like effect in NRK-49F cells and induced TMEM45A and Jagged1/Notch expression. Collectively, these results demonstrate that TMEM45A plays an important role in renal fibrosis by regulating ECM components and Jagged1/Notch pathway.

Abstract 17015: PGC-1alpha/Arhgap6 Axis Mediates Diabetic Vascular Dysfunction Through Diminution of Endothelial Glycolytic Flux

Introduction: Aberrant angiogenesis in diabetes (DM) is caused by failure of local endothelial cells (ECs) to properly undergo VEGF-driven migration, and leads to a high propensity to develop critical limb ischemia. We recently showed that type 1 and 2 DM induce PGC-1α in ECs, which in turn strongly blocks EC migration and angiogenesis. Despite this therapeutic potential, PGC-1α may not be “druggable” due to the diverse distribution and functions in non-vascular tissues. Thus, understanding EC PGC-1α effectors is critical, which may involve bioenergetics since PGC-1α broadly induces mitochondrial OxPhos. Although ECs show high rate of glycolytic flux and synthesis of lactate, a burgeoning angiogenic mediator, this is blunted in DM via mechanisms still ill-defined. We hypothesized that a novel PGC-1α target in DM mediates blunted EC glycolysis and migration. Methods and Results: By leveraging NRF1/2-binding defective PGC-1α, we show that PGC-1α blocks glycolysis, through PPARgamma-driven Notch induction independent of mitochondrial activation, which fully accounts for PGC-1α inhibition of EC migration in DM. mRNA microarray identified ARHGAP6, a Rho-GAP and actin cytoskeletal modulator, as a DM-inducible main effector of PGC-1α axis to inhibit EC lactate synthesis and migration. Data suggest the causal role for ARHGAP6 in the reduced migration and glycolysis (Seahorse XF analyzer) of STZ-induced DM mouse ECs. Impaired migration of DM ECs and ECs overexpressing PPARgamma or ARHGAP6 was associated with decreased activity of Rac1 and rescued by addition of lactate. Reduced GTP-Rac1 in ECs overexpressing PGC-1α was restored by knockdown of ARHGAP6. Positive feedback loop between Rac1 and LDHA was demonstrated, suggesting a cytoskeletal-metabolic module to mediate EC migration and dysfunction. Conclusions: PGC-1α/ ARHGAP6 inhibition of EC glycolysis and lactate synthesis is a key mechanism of impaired EC migration in DM angiopathy. Our data provide important insights into the mechanistic link between EC actin cytoskeleton and bioenergetics mediated by ARHGAP6, and opportunities to restore diminished glycolysis in DM ECs by targeting PGC-1α axis component ARHGAP6 to develop safe and efficacious therapeutic since it is dispensable for health.

Serelaxin induces Notch1 signaling and alleviates hepatocellular damage in orthotopic liver transplantation.

Liver ischemia-reperfusion injury (IRI) represents a risk factor for early graft dysfunction and an obstacle to expanding donor pool in orthotopic liver transplantation (OLT). We have reported on the crucial role of macrophage Notch1 signaling in mouse warm hepatic IRI model. However, its clinical relevance or therapeutic potential remain unknown. Here, we used Serelaxin (SER), to verify Notch1 induction and putative hepatoprotective function in ischemia-reperfusion-stressed OLT. C57BL/6 mouse livers subjected to extended (18-hour) cold storage were transplanted to syngeneic recipients. SER treatment at reperfusion ameliorated IRI, improved post-OLT survival, decreased neutrophil/macrophage infiltration, and suppressed proinflammatory cytokine programs, while simultaneously increasing Notch intracellular domain (NICD) and hairy and enhancer of split 1 (Hes1) target genes. In bone marrow-derived macrophage cultures, SER suppressed proinflammatory while enhancing antiinflammatory gene expression concomitantly with increased NICD and Hes1. Hepatic biopsies from 21 adult primary liver transplant patients (2hours postreperfusion) were divided into low-NICD (n=11) and high-NICD (n=10) expression groups (western blots). Consistent with our murine findings, human livers characterized by high NICD were relatively IRI resistant, as shown by serum alanine aminotransferase (ALT) levels at day 1 post-OLT. Our study documents the efficacy of SER-Notch1 signaling in mouse OLT and highlights the protective function of Notch1 in liver transplant patients.

Effect of Ilexonin A on the Notch signaling pathway following cerebral ischemia-reperfusion in rats

Ilexonin A is a pentacyclic triterpenes, extracted from the herb Ilex pubescens with anti-thrombosis and anti-inflammatory activity, and improves blood circulation. This study determines whether Ilexonin A (IA) activates Notch signaling to induce neurogenesis following cerebral ischemia-reperfusion injury. We investigated the effect of Ilexonfound in the Ilexonin A-treatedin A on post-ischemic proliferation and differentiation of neural stem cells. Male Sprague-Dawley rats were divided into four groups: normal (n=6), sham-operated (n=6), control (n=24), and IA-treated (n=24). Focal cerebral ischemia-reperfusion injury was induced by the Middle Cerebral Artery Occlusion (MCAO) method. Ilexonin A at dose of 40 mg/kg was administered via intraperitoneal injection immediately following MCAO and then once daily until the rats were sacrificed. 5-bromo 2-deoxyuridine labeled proliferating cells and neurogenesis and Notch induction in the ischemic tissue perienchyma were observed at d 1, 3, 7, and 14 following ischemia by immunohistochemistry. NICD protein, Notch1, PS1, and HES1 gene expression was detected. Ilexonin A decreased the infarct volume and improved neurological deficits in a time-dependent manner. A higher number of Brdu/Nestin double-positive cells was found in the Ilexonin A-treated rats, compared with the rats exposed to ischemia only at both 3 d (14.33 ± 1.63 vs. 11.33 ± 1.21, *p<0.05) and 7 d (29.33 ± 2.58 vs. 24.50 ± 2.7, *p<0.05). Ilexonin A activates Notch signaling by increasing the expression of NICD at both 3 d (75.83 ± 1.94 vs. 69.67 ± 3.93, #p<0.01) and 7 d (53.67 ± 2.42 vs. 50.17 ± 2.93, #p<0.01). Ilexonin A also increased the gene expression levels of Notch1, PS1, and HES1 at 3 d after reperfusion in the rats. These findings suggested that the protective effects of Ilexonin A were associated with neuronal regeneration, and the inactivation of Notch signaling pathways.

Bmp2 and Notch cooperate to pattern the embryonic endocardium.

Signaling interactions between the myocardium and endocardium pattern embryonic cardiac regions, instructing their development to fulfill specific functions in the mature heart. We show that ectopic Bmp2 expression in the mouse chamber myocardium changes the transcriptional signature of adjacent chamber endocardial cells into valve tissue, and enables them to undergo epithelial-mesenchyme transition. This induction is independent of valve myocardium specification and requires high levels of Notch1 activity. Biochemical experiments suggest that Bmp2-mediated Notch1 induction is achieved through transcriptional activation of the Notch ligand Jag1, and physical interaction of Smad1/5 with the intracellular domain of the Notch1 receptor. Thus, widespread myocardial Bmp2 and endocardial Notch signaling drive presumptive ventricular endocardium to differentiate into valve endocardium. Understanding the molecular basis of valve development is instrumental to designing therapeutic strategies for congenital heart valve defects.

Notch3 as a novel therapeutic target in metastatic medullary thyroid cancer

Medullary thyroid cancer portends poor survival once liver metastasis occurs. We hypothesize that Notch3 overexpression in medullary thyroid cancer liver metastasis will decrease proliferation and growth of the tumor. TT cells were modified genetically to overexpress Notch3 in the presence of doxycycline, creating the TT-Notch3 cell line. Mice were injected intrasplenically with either TT-Notch3 or control vector TT-TRE cells. Each cell line had 3 treatment groups: control with 12 weeks of standard chow, early DOX with doxycycline chow at day 0 and for 70 days thereafter, and late DOX with doxycycline chow at 8 weeks. Each animal underwent micro-computed tomography to evaluate for tumor formation and tumor quantification was performed. Animals were killed at 12 weeks, and the harvested liver was stained with Ki-67, hematoxylin and eosin, and Notch3. Induction of Notch3 did not prevent formation of medullary thyroid cancer liver metastases as all mice in the early DOX group developed tumors. However, induction of Notch after medullary thyroid cancer liver tumor formation decreased tumor size, as seen on micro-computed tomography scans (late DOX group). This translated to a 37-fold decrease in tumor volume (P = .001). Notch3 overexpression also resulted in decreased Ki-67 index (P = .038). Moreover, Notch3 induction led to increased areas of neutrophil infiltration and necrosis on hematoxylin and eosin staining of the tumors CONCLUSION: Notch3 overexpression demonstrates an antiproliferative effect on established metastatic medullary thyroid cancer liver tumors and is a potential therapeutic target in treatment.

Antineoplastic effects of histone deacetylase inhibitors in neuroendocrine cancer cells are mediated through transcriptional regulation of Notch1 by activator protein 1.

Notch signaling is minimally active in neuroendocrine (NE) cancer cells. While histone deacetylase inhibitors (HDACi) suppress NE cancer growth by inducing Notch, the molecular mechanism underlying this interplay has not yet been defined. NE cancer cell lines BON, H727, and MZ‐CRC‐1 were treated with known HDACi Thailadepsin‐A (TDP‐A) and valproic acid (VPA), and Notch1 mRNA expression was measured with RT‐PCR. Truncated genomic fragments of the Notch1 promotor region fused with luciferase reporter were used to identify the potential transcription factor (TF) binding site. The key regulatory TF was identified with the electrophoretic mobility shift assay (EMSA). The effect of HDACi on Notch1 level was determined before and after silencing the TF. TDP‐A and VPA induced Notch1 mRNA in a dose‐dependent manner. A functional DNA motif at −80 to −52 from the Notch1 start codon responsible for the HDACi‐dependent Notch1 induction was identified. Mutation of this core sequence failed to induce luciferase activity despite HDACi treatment. EMSA showed the greatest gel shift with AP‐1 in nuclear extracts. Knockdown of AP‐1 significantly attenuated the effect of HDACi on Notch1 induction. Interestingly, AP‐1 transfection did not alter Notch1 level, suggesting that AP‐1 is necessary but insufficient for HDACi activation of Notch1. Therefore, AP‐1 is the TF that binds to a specific transcription‐binding site within the Notch1 promotor region to trigger Notch1 transcription. Elucidating the HDACi activation mechanism may lead to the development of novel therapeutic options against NE cancers and facilitate the identification of clinical responders and prevent adverse effects.

Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence.

During development, hematopoietic stem cells (HSCs) emerge from aortic endothelial cells (ECs) through an intermediate stage called hemogenic endothelium by a process known as endothelial-to-hematopoietic transition (EHT). While Notch signaling, including its upstream regulator Vegf, is known to regulate this process, the precise molecular control and temporal specificity of Notch activity remain unclear. Here, we identify the zebrafish transcriptional regulator evi1 as critically required for Notch-mediated EHT Invivo live imaging studies indicate that evi1 suppression impairs EC progression to hematopoietic fate and therefore HSC emergence. evi1 is expressed in ECs and induces these effects cell autonomously by activating Notch via pAKT Global or endothelial-specific induction of notch, vegf, or pAKT can restore endothelial Notch and HSC formations in evi1 morphants. Significantly, evi1 overexpression induces Notch independently of Vegf and rescues HSC numbers in embryos treated with a Vegf inhibitor. In sum, our results unravel evi1-pAKT as a novel molecular pathway that, in conjunction with the shh-vegf axis, is essential for activation of Notch signaling in VDA endothelial cells and their subsequent conversion to HSCs.

Coronary Microvascular Smooth Muscle‐Endothelial Cell Communication and Notch 3 Signaling in Type 2 Diabetes

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. The current study tested the hypothesis that VSMC‐endothelial cell (EC) Notch 3/Jagged 1 signaling is aberrant to account for adverse remodeling and stiffness observed in type 2 diabetic coronary resistance microvessels (CRMs). Low‐passage primary coronary VMSCs were isolated from normal and type 2 diabetic db/db mice and were either mono‐ or co‐cultured with normal or type 2 diabetic human coronary microvascular endothelial cells (hCMECs), respectively (n=4–6 per group). In separate experiments, normal (n=3) and diabetic db/db CRMs (n=3) were isolated, mounted, and perfusion pressure fixed on a pressure myograph. The size and frequency of fenestrae in the internal elastic laminas of intact CRMs were assessed using Alexa Fluor 633 hydrazide on a confocal microscope. Coronary VSMCs isolated from db/db mice expressed higher Notch 3 mRNA (2.12±0.10 vs. 1.00±0.20; p&lt;0.01) compared to normal. Co‐culture of coronary VSCMs with hCMECs caused a robust induction of Notch 3 expression in normal VSMCs (3.67±0.60 vs. 1.00, p&lt;0.001), which was exacerbated in coronary VSMCs isolated from db/db mice (5.52±0.51 vs. 3.67±0.60, p&lt;0.05). Alpha‐smooth muscle actin (α‐SMA) trended lower in db/db coronary VSMCs (p=0.07), while co‐culture with hCMECs did not alter α‐SMA expression. Diabetic hCMECs expressed lower Jagged 1 mRNA compared to normal (0.58±0.04 vs. 1.00±0.07; p&lt;0.01); co‐culture of hCMECs and coronary VSMCs caused a robust reduction in Jagged 1 expression in normal cells (0.63±0.06 vs. 1.00±0.07, p&lt;0.001), while no further reduction was observed in co‐cultured diabetic cells. The number of fenestrae in the internal elastic lamina was reduced in diabetic CRMs (0.028±0.005 vs. 0.040±0.001 fen/μm2, p&lt;0.05), while the size of the fenestrae remained unchanged. These data show that Notch3/Jagged 1 expression and VSMC differentiation are altered in diabetic coronary resistance microvessel cells, and VSMC‐EC contact via co‐culture induces differential dysregulation of Notch3/Jagged1 signaling. The data further show a reduction in the number of fenestrae in db/db CRMs, suggesting that the opportunity for Notch3/Jagged1 signaling to occur in vivo may be reduced to contribute to adverse remodeling and stiffness observed in those blood vessels.Support or Funding InformationAHA 15UFEL22760002 to MK; NIH K99HL116769 and NCH to AJT.

Dedifferentiation of patient-derived glioblastoma multiforme cell lines results in a cancer stem cell-like state with mitogen-independent growth.

Emerging evidence shows that glioblastoma multiforme (GBM) originates from cancer stem cells (CSCs). Characterization of CSC-specific signalling pathways would help identify new therapeutic targets and perhaps lead to the development of more efficient therapies selectively targeting CSCs. Here; we successfully dedifferentiated two patient-derived GBM cell lines into CSC-like cells (induced glioma stem cells, iGSCs) through expression of Oct4, Sox2 and Nanog transcription factors. Transformed cells exhibited significant suppression of epidermal growth factor receptor and its downstream pathways. Compared with parental GBM cells, iGSCs formed large neurospheres even in the absence of exogenous mitogens; they exhibited significant sensitivity to salinomycin and chemoresistance to temozolomide. Further characterization of iGSCs revealed induction of NOTCH1 and Wnt/β-catenin signalling and expression of CD133, CD44 and ALDH1A1. Our results indicate that iGSCs may help us understand CSC physiology and lead to development of potential therapeutic interventions aimed at differentiating tumour cells to render them more sensitive to chemotherapy or other standard agents.

Notch1 acts via Foxc2 to promote definitive hematopoiesis via effects on hemogenic endothelium

AbstractHematopoietic and vascular development share many common features, including cell surface markers and sites of origin. Recent lineage-tracing studies have established that definitive hematopoietic stem and progenitor cells arise from vascular endothelial–cadherin+ hemogenic endothelial cells of the aorta-gonad-mesonephros region, but the genetic programs underlying the specification of hemogenic endothelial cells remain poorly defined. Here, we discovered that Notch induction enhances hematopoietic potential and promotes the specification of hemogenic endothelium in differentiating cultures of mouse embryonic stem cells, and we identified Foxc2 as a highly upregulated transcript in the hemogenic endothelial population. Studies in zebrafish and mouse embryos revealed that Foxc2 and its orthologs are required for the proper development of definitive hematopoiesis and function downstream of Notch signaling in the hemogenic endothelium. These data establish a pathway linking Notch signaling to Foxc2 in hemogenic endothelial cells to promote definitive hematopoiesis.

Tumor-suppressor role of Notch3 in medullary thyroid carcinoma revealed by genetic and pharmacological induction.

Notch1-3 are transmembrane receptors that appear to be absent in medullary thyroid cancer (MTC). Previous research has shown that induction of Notch1 has a tumor-suppressor effect in MTC cell lines, but little is known about the biologic consequences of Notch3 activation for the progression of the disease. We elucidate the role of Notch3 in MTC by genetic (doxycycline-inducible Notch3 intracellular domain) and pharmacologic [AB3, novel histone deacetylase (HDAC) inhibitor] approaches. We find that overexpression of Notch3 leads to the dose-dependent reduction of neuroendocrine tumor markers. In addition, Notch3 activity is required to suppress MTC cell proliferation, and the extent of growth repression depends on the amount of Notch3 protein expressed. Moreover, activation of Notch3 induces apoptosis. The translational significance of this finding is highlighted by our observation that MTC tumors lack active Notch3 protein and reinstitution of this isoform could be a therapeutic strategy to treat patients with MTC. We demonstrate, for the first time, that overexpression of Notch3 in MTC cells can alter malignant neuroendocrine phenotype in both in vitro and in vivo models. In addition, our study provides a strong rationale for using Notch3 as a therapeutic target to provide novel pharmacologic treatment options for MTC.

Abstract 1149: The role of the novel Notch1-Sox9 signaling axis in NSCLC progression and EMT

Abstract Sox9 plays critical roles in the specification and differentiation of numerous progenitor and differentiated cell types during embryonic and fetal development. Sox9 is overexpressed in 40 - 50% of lung adenocarcinomas and associated with poor prognosis in lung cancer patients. We set out to identify upstream pathways that regulate Sox9 expression in lung cancer, as well as the role of Sox9 in lung adenocarcinoma progression. Several developmental and stem cell pathways are known to induce Sox9 transcription during carcinogenesis, including the TGB-β, Wnt/β-catenin, Sonic Hedgehog, and NF-κB signaling. Sox9 has also been shown to be a transcriptional target of the Notch pathway during mouse development, although the binding sites for Notch within the mouse Sox9 promoter are not conserved in humans. We mined gene expression data from three publicly available datasets and found that Hes1, a known Notch target gene, is co-expressed with Sox9 in lung adenocarcinoma. Furthermore, Sox9 mRNA and protein levels were upregulated over 100-fold as early as 14 days after Notch1 induction in the Notch1-induced mouse model of lung cancer, suggesting that Sox9 overexpression is an early event during lung cancer development. Through a series of in vitro assays, we determined that Sox9 is downstream of Notch1 in lung adenocarcinoma cell lines. By ChIP we determined that Sox9 is a direct target of Notch1 and using luciferase reporter assays, we located the previously unidentified human RBP-Jκ binding site, the principle effector of canonical Notch1 signaling, immediately upstream of the Sox9 transcriptional start site. We also examined TGF-β, a known inducer of epithelial-to-mesenchymal transition (EMT) in lung cancer. We determined that induction of Sox9 expression by Notch1 is independent of TGF-β signaling and that TGF-β and Notch1 cooperate in their regulation of Sox9 expression. Loss of Notch1 expression led to an induced MET phenotype, characterized by decreased cell invasion/migration, MET-like morphological changes, and increased E-cadherin expression, which were rescued by Sox9 overexpression. Our data also demonstrate that Sox9 contributes to Notch1-induced EMT in lung adenocarcinoma. These results establish Sox9 as a key Notch1 target gene mediating Notch1-induced EMT independent of TGF-β, leading to poor survival in lung adenocarcinoma. Citation Format: Kathleen M. Capaccione, Xuehui Hong, Katherine M. Morgan, Thaddeus D. Allen, Gregory D. Miles, Elke K. Markert, J. Michael Bishop, Sharon R. Pine. The role of the novel Notch1-Sox9 signaling axis in NSCLC progression and EMT. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1149. doi:10.1158/1538-7445.AM2014-1149

Abstract B17: Sox9 mediates Notch pathway-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma.

Abstract Background: Sox9 plays critical roles in the specification and differentiation of numerous progenitor and differentiated cell types during embryonic and fetal development. Sox9 is overexpressed in 40 - 50% of lung adenocarcinomas and associated with poor prognosis in lung cancer patients. We set out to identify the upstream pathways that regulate Sox9 expression in lung cancer as well as the role of Sox9 in lung adenocarcinoma progression. Several developmental and stem cell pathways are known induce Sox9 transcription during carcinogenesis, including the TGB-β, Wnt/β-catenin, Sonic Hedgehog, and NF-κB signaling pathways. SOX9 has also been shown to be a transcriptional target of the Notch pathway during mouse development, although the binding sites for Notch within the mouse SOX9 promoter are not conserved in humans. Results: We mined gene expression data from three publicly available datasets and found that Hes1, a known Notch target gene, is co-expressed with Sox9 in lung adenocarcinoma. We validated the data by identifying significant overlap in Sox9 and Hes1 protein expression levels in a human lung cancer tissue microarray. Furthermore, Sox9 mRNA and protein levels were upregulated over 100-fold as early as 14 days after Notch1 induction in the Notch1-induced mouse model of lung cancer, suggesting that Sox9 overexpression is an early event during lung cancer development. Through a series of in vitro assays, we determined that Sox9 is immediately downstream of Notch1, but not Notch3, in lung adenocarcinoma cell lines. By ChIP and luciferase reporter assays, we located the previously unidentified human RBPjk binding site, the principle effector of canonical Notch1 signaling, immediately upstream of the SOX9 transcriptional start site. We also examined TGF-β, a known inducer of epithelial-to-mesenchymal transition (EMT) in lung cancer. We determined that induction of Sox9 expression by Notch1 is independent of TGF-β signaling, that TGF-β upregulates both Sox9 and Notch1 expression, and that TGF-β and Notch1 cooperate in their regulation of Sox9 expression. Sox9 overexpression led to an induced EMT phenotype, characterized by increased cell invasion/migration, and EMT-related gene expression changes. Sox9 mRNA expression correlated with EMT-like gene expression signatures in several gene expression microarray datasets, and Sox9 protein levels were negatively correlated with expression of the epithelial marker, E-cadherin, in a lung cancer tissue microarray. Our data also demonstrate that Notch1-induced EMT in lung adenocarcinoma is mediated, at least partly, through Sox9. Conclusion: These results establish Sox9 as a key Notch1 target gene mediating Notch1- and TGF-β- induced EMT, leading to poor survival in lung adenocarcinoma. Citation Format: Kathleen Capaccione, Xuehui Hong, Katherine M. Morgan, Wenyu Liu, Thaddeus Allen, J. Michael Bishop, Sharon R. Pine. Sox9 mediates Notch pathway-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B17.