Notch1 shRNA Research Articles

Notch1 hyperactivity drives ubiquitination of NOX2 and dysfunction of CD8+ regulatory T cells in patients with systemic lupus erythematosus.

Patients with systemic lupus erythematosus (SLE) display heightened immune activation and elevated IgG autoantibody levels, indicating compromised regulatory T cell (Tregs) function. Our recent findings pinpoint CD8+ Tregs as crucial regulators within secondary lymphoid organs, operating in a NOX2-dependent mechanism. However, the specific involvement of CD8+ Tregs in SLE pathogenesis and the mechanisms underlying their role remain uncertain. SLE and healthy individuals were enlisted to assess the quantity and efficacy of Tregs. CD8+CD45RA+CCR7+ Tregs were generated ex vivo, and their suppressive capability was gauged by measuring pZAP70 levels in targeted T cells. Notch1 activity was evaluated by examining activated Notch1 and HES1, with manipulation of Notch1 accomplished with Notch inhibitor DAPT, Notch1 shRNA, and Notch1-ICD. To create humanized SLE chimeras, immune-deficient NSG mice were engrafted with PBMCs from SLE patients. We observed a reduced frequency and impaired functionality of CD8+ Tregs in SLE patients. There was a downregulation of NOX2 in CD8+ Tregs from SLE patients, leading to a dysfunction. Mechanistically, the reduction of NOX2 in SLE CD8+ Tregs occurred at a post-translational level rather than at the transcriptional level. SLE CD8+ Tregs exhibited heightened Notch1 activity, resulting in increased expression of STUB1, an E3 ubiquitin ligase that binds to NOX2 and facilitates its ubiquitination. Consequently, restoring NOX2 levels and inhibiting Notch1 activity could alleviate the severity of the disease in humanized SLE chimeras. Notch1 is the cell-intrinsic mechanism underlying NOX2 deficiency and CD8+ Treg dysfunction, serving as a therapeutic target for clinical management of SLE.

Limb Remote Ischemic Conditioning Promotes Neurogenesis after Cerebral Ischemia by Modulating miR-449b/Notch1 Pathway in Mice.

Neurogenesis plays an important role in the prognosis of stroke patients and is known to be promoted by the activation of the Notch1 signaling pathway. Studies on the airway epithelium have shown that miR-449b represses the Notch pathway. The study aimed to investigate whether limb remote ischemic conditioning (LRIC) was able to promote neurogenesis in cerebral ischemic mice, and to investigate the role of the miR-449b/Notch1 pathway in LRIC-induced neuroprotection. Male C57BL/6 mice (22–25 g) were subjected to transient middle cerebral artery occlusion (MCAO), and LRIC was performed in the bilateral lower limbs immediately after MCA occlusion. Immunofluorescence staining was performed to assess neurogenesis. The cell line NE-4C was used to elucidate the proliferation of neuronal stem cells in 8% O2. After LRIC treatment on day 28, mice recovered neurological function. Neuronal precursor proliferation was enhanced in the SVZ, and neuronal precursor migration was enhanced in the basal ganglia on day 7. LRIC promoted the improvement of neurological function in mice on day 28, promoted neuronal precursor proliferation in the SVZ, and enhanced neuronal precursor migration in the basal ganglia on day 7. The neurological function score was negatively correlated with the number of BrdU-positive/DCX-positive cells in the SVZ and striatum. LRIC promoted activated Notch1 protein expression in the SVZ and substantially downregulated miR-449b levels in the SVZ and plasma. In vitro, miR-449b was found to target Notch1. Lentivirus-mediated miR-449b knockdown increased Notch1 levels in NE-4C cells and increased proliferation in the cells. The effects of miR-449b inhibition on neurogenesis were ablated by the application of Notch1 shRNA. Our study showed that LRIC promoted the proliferation and migration of neural stem cells after MCAO, and these effects were modulated by the miR-449b/Notch1 pathway.

Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Alleviate Diffuse Alveolar Hemorrhage Associated with Systemic Lupus Erythematosus in Mice by Promoting M2 Macrophage Polarization via the microRNA-146a-5p/NOTCH1 Axis

ABSTRACT Systemic lupus erythematosus (SLE)-associated diffuse alveolar hemorrhage (DAH) is a rare but extremely harmful condition. The current study sought to dissect the mechanisms underlying the effects of human umbilical cord mesenchymal stem cell (HUCMSC)-derived exosomes on M2 macrophage polarization in SLE-associated DAH via the microRNA (miR)-146a-5p/NOTCH1 axis. A DAH mouse model was established using pristane. Exosomes were isolated from HUCMSCs transfected or untransfected with the miR-146a-5p antagonist or agonist and their NCs and then injected into DAH mice. Additionally, miR-146a-5p was overexpressed in macrophages. Expression of miR-146a-5p, NOTCH1, M1 macrophage markers, and M2 macrophage markers was measured in mice and macrophages, and inflammatory factor levels were detected. Mouse lung injuries were evaluated, so was the binding of miR-146a-5p to NOTCH1. Rescue experiments were conducted in mice and macrophages using NOTCH1 shRNA and pcDNA3.1-NOTCH1, respectively. NOTCH1 expression was enhanced in DAH mice. HUCMSC-derived exosomes reduced NOTCH1 expression, bleeding, inflammation, and M1 macrophage polarization but elevated M2 macrophage polarization in lung tissues of DAH mice. Mechanistically, NOTCH1 is negatively targeted by miR-146a-5p. miR-146a-5p overexpression diminished M1 marker and inflammatory factor levels but enhanced M2 marker levels in macrophages, which was nullified by NOTCH1 overexpression. HUCMSC-derived exosomes with miR-146a-5p inhibition increased NOTCH1 expression, worsened bleeding and inflammation, and augmented M1 macrophage polarization while decreasing M2 macrophage polarization in lung tissues of DAH mice, which was abrogated by silencing NOTCH1. HUCMSC-derived exosomes diminished NOTCH1 expression to accelerate M2 macrophage polarization via delivery of miR-146a-5p, thus alleviating SLE-associated DAH in mice.

Abstract 81: Trop2 regulates prostate cancer growth and metastasis through distinct molecular mechanisms

Abstract Background: The first line of treatment for men with advanced prostate cancer is androgen deprivation therapy. Although initial responses are observed, prostate cancer commonly relapses in its lethal metastatic form referred to as castration resistant prostate cancer (CRPC) with 1-2 years mean survival time. Neuroendocrine prostate cancer (NEPC) is highly aggressive, AR independent subtype and usually emerges post castration resistance. We recently identified that the cell surface receptor, Trop2, is a new driver of NEPC. Moreover, we demonstrated that Trop2 regulates prostate cancer growth and metastasis. In this study, we set out to delineate the molecular mechanisms through which Trop2 regulates prostate cancer growth and metastasis. Methods: Proximity-dependent Biotin Identification (BioID) followed by mass spectrometry was performed to identify Trop2 interactome. Lentiviral infection was used to generate prostate cancer cell lines with over-expression of Trop2 and knock-down of NOTCH1, SLC4A7, PLEC, and OCLN shRNA to modulate gene expression levels. In vitro functional assays were performed including colony formation, and Matrigel drop 3D cell invasion assays. Results: The Trop2 membrane interactome was identified utilizing Proximity-dependent Biotin Identification (BioID) in living cells and uncovered that Trop2-mediated prostate cancer growth and metastasis are orchestrated by distinct downstream pathways including Notch signaling (NOTCH1), control of intracellular pH (SLC4A7), exosome secretion (PLEC), and tight junctions (OCLN). Interaction of Trop2 binding partners including NOTCH1, SLC4A7, PLEC and OCLN with Trop2 were further validated by fluorescence resonance energy transfer (FRET) using confocal microscopy. Moreover, knocking down the Trop2 interacting partners in Trop2 over-expressing prostate cancer cells suppressed Trop2-driven growth and invasion ability. Conclusions: In a previous study, we identified cell surface receptor, Trop2, as a novel driver of metastatic NEPC. Herein, our new findings reveal that Trop2 interacts with NOTCH1, SLC4A7, PLEC, and OCLN, which may highlight novel biological functions of Trop2 in prostate tumorigenesis and provide new understanding of the potential mechanism of neuroendocrine differentiation and metastasis to provide new therapeutic strategy for metastatic CRPC with neuroendocrine features. Citation Format: Tanya Ivanova Stoyanova, En-Chi Hsu, Shiqin Liu, Fernando Jose Garcia Marques, Abel Bermudez, Merve Aslan, Michelle Shen, Sharon Pitteri, James D. Brooks. Trop2 regulates prostate cancer growth and metastasis through distinct molecular mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 81.

Trop2 regulates prostate cancer growth and metastasis through distinct molecular mechanisms

Background Prostate cancer is the most common non-cutaneous cancer and second leading cause of cancer related deaths in men in the United States. The first line of treatment for men with advanced prostate cancer is androgen deprivation therapy. Although initial responses are observed, prostate cancer commonly relapses in its lethal metastatic form referred to as castration resistant prostate cancer (CRPC) with 1-2 years mean survival time. Neuroendocrine prostate cancer (NEPC) is highly aggressive, AR independent subtype and usually emerges post castration resistance. We recently identified that the cell surface receptor, Trop2, is a new driver of NEPC. Moreover, we demonstrated that Trop2 regulates prostate cancer growth and metastasis. In this study, we set out to delineate the molecular mechanisms through which Trop2 regulates prostate cancer growth and metastasis. Methods Proximity-dependent Biotin Identification (BioID) followed by mass spectrometry was performed to identify Trop2 interactome. Lentiviral infection was used to generate prostate cancer cell lines with over-expression of Trop2 and knock-down of NOTCH1, SLC4A7, PLEC, and OCLN shRNA to modulate gene expression levels.In vitro functional assays were performed includingcolony formation, and Matrigel drop 3D cell invasion assays. Results The Trop2 membrane interactome was identified utilizing Proximity-dependent Biotin Identification (BioID) in living cells and uncovered that Trop2-mediated prostate cancer growth and metastasis are orchestrated by distinct downstream pathways including Notch signaling (NOTCH1), control of intracellular pH (SLC4A7), exosome secretion (PLEC), and tight junctions (OCLN). Interaction of Trop2 binding partners including NOTCH1, SLC4A7, PLEC and OCLN with Trop2 were further validated by fluorescence resonance energy transfer (FRET) using confocal microscopy. Moreover, knocking down the Trop2 interacting partners in Trop2 over-expressing prostate cancer cells suppressed Trop2-driven growth and invasion ability. Conclusions In a previous study, we identified cell surface receptor, Trop2, as a novel driver of metastatic NEPC. Herein, our new findings reveal that Trop2 interacts with NOTCH1, SLC4A7, PLEC, and OCLN, which may highlight novel biological functions of Trop2 in prostate tumorigenesis and provide new understanding of the potential mechanism of neuroendocrine differentiation and metastasis to provide new therapeutic strategy for metastatic CRPC with neuroendocrine features.

Effects of targeted Notch1 silencing on the biological processes of the T24 and 5637 cells in vitro.

The present study aimed to investigate the roles of Notch1 in the biological processes of bladder cancer cells (BCCs) in vitro. Short hairpin (sh)RNA targeting Notch1 was designed and constructed, and the T24 and 5637 BCCs were selected for transfection. The cells were classified into two groups: shRNA negative control (NC) and Notch1 shRNA. MTT and Transwell assays, and flow cytometry were performed to examine the changes in cell proliferation, invasiveness, and apoptosis, respectively. In addition, reverse transcription-quantitative PCR and western blot analysis was used to detect the mRNA and protein expression levels of apoptosis-related proteins (Bax, Bid and Bcl2) and epithelial-mesenchymal transition factors (vimentin and E- and N-cadherin). Compared with that in the shRNA NC group, the Notch1 shRNA group showed significantly decreased cell proliferation rate and invasiveness; increased apoptotic rate; elevated mRNA expression levels of Bad, Bid and E-cadherin; and reduced mRNA expression levels of Bcl2, N-cadherin and vimentin. The trends for protein expression levels were the same as those for mRNA levels. Notch1 silencing inhibited invasion and promoted apoptosis of BCCs.

Abstract 3124: SNAI2 function in embryonal RMS

Abstract Rhabdomyosarcoma (RMS) is a pediatric malignancy of the muscle and a key feature of the histology is muscle cells blocked in differentiation despite robust expression of diagnostic muscle differentiation factors MYOD1 and Myogenin. Thus, there are mechanisms operating in tumors that block myogenic differentiation. We previously defined roles in differentiation, self-renewal and growth for a NOTCH1/SNAI1/MEF2C pathway in Embryonal RMS, the major RMS subtype driven predominantly by Ras signaling. However, we observed that SNAI1 knockdown did not result in as robust differentiation as in NOTCH1 shRNA knockdown cells. We hypothesized that SNAI1 and SNAI2 function might be redundant in ERMS. Analysis of SNAI2 expression in primary tumors and cell lines finds that indeed SNAI2 is highly expressed in RMS and ERMS tumors typically express higher SNAI2 compared to SNAI1. To address SNAI2 function, we knocked down SNAI2 using 2 independent shRNAs and assessed effects on differentiation, self-renewal and growth in ERMS RD, SMS-CTR and JR1 cells. Knockdown of SNAI2 both in stable and transient experiments resulted in robust differentiation (10 fold increase) as assessed by differentiated myosin MF20 expression in RD, JR1 and SMS-CTR cells p<0.001). This increase in differentiation was associated with increased expression differentiation genes including MYOD1, MYOGENIN, MEF2C, MEF2D and differentiated myosins and a loss of precursor gene PAX7 as assessed by quantitative RT-PCR and protein expression. SNAI2 knockdown RD and JR1 cells also formed significantly fewer rhabdospheres (p<0.01). Finally, SNAI2 knockdown with 2 independent shRNAs resulted in significantly smaller and more differentiated tumors when xenografted subcutaneously in vivo in SCID mice. Since SNAI2 is a known DNA binding transcriptional repressor, we performed ChIPseq for SNAI2 and H3K27acetyl in SMS-CTR and RD cells coupled with RNAseq to define direct and indirect SNAI2 regulated genes. Our ChIPseq results identified the known SNAI2 DNA binding motif, however additionally we find that SNAI2 chromatin binding significantly enriched for myogenic E box elements bound by MYOD1, E2A and Myogenin. Additionally, SNAI2 binding was more significantly associated with the muscle differentiation program. Given the differential roles of MYOD1 and SNAI2 on gene activation vs. gene repression, we hypothesized that SNAI2 by competing with MYOD1 at terminally differentiated genes maintains early cell cycle effects of MYOD1 but blocks terminal differentiation. Analysis of MYOD1 expression in SNAI2 knockdown cells finds a redistribution of MYOD1 binding from cell cycle to more differentiated muscle genes and is associated with a concomitant exit from the cell cycle and robust differentiation. In summary, SNAI2 is a robust driver of ERMS differentiation and in vivo growth. High SNAI2 expression competes with MYOD1 at terminally differentiated genes blocking differentiation and exit from the cell cycle in ERMS. Citation Format: Silvia Pomelo, Prethish Sreenivas, Berkley Gryder, Long Wang, Baxi Kunal, Nicole Hensch, Eleanor Chen, Peter Houghton, Rossella Rota, Javed Khan, Myron S. Ignatius. SNAI2 function in embryonal RMS [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 3124.

PF581 FUNCTIONAL HIGH‐THROUGHPUT SCREENING FOR IDENTIFICATION OF NOTCH1 DOWNSTREAM EFFECTORS AS NOVEL THERAPEUTIC TARGETS IN MULTIPLE MYELOMA

Background:Intrinsic and acquired drug resistance of multiple myeloma (MM) cells often cause disease progression in MM patients. Therefore, the identification of novel drug targets is needed to further improve treatment strategies. The deregulated Notch1 pathway is a master regulator of MM onset, progression and drug resistance. Inhibition of Notch through use of γ‐secretase inhibitors causes serious side effects and is not used in the clinic for treatment of MM patients.Aims:In this study, we identify novel druggable downstream effectors of the Notch1 pathway to overcome drug resistance of MM cells.Methods:We employ high‐throughput (HT) functional screening based on tailored Notch1 shRNA libraries under selective pressure of chemotherapeutic agents. First, we performed genome‐wide expression profiling through RNA sequencing (RNAseq) in Notch‐positive MM.1S cells after inhibition of the pathway. Strongly and consistently regulated genes (log2FC; padj <0.05) were chosen as targets for two tailored shRNA libraries. Functional screens are conducted using native U266 cells (positive selection screens) or U266 cells engineered to express the Notch1 variants NIC or NΔE (negative selection screens). NIC is the active and γ‐secretase‐independent form of Notch1. By using the membrane‐bound and γ‐secretase‐dependent NΔE variant, screening additionally comprises investigation of cooperation between Notch1 and cytoplasmic signaling complexes. Following transduction of U266 cells with the corresponding shRNA library, cells are cultured under selective pressure of Melphalan, Bortezomib or Lenalidomide. HT sequencing and bioinformatics analysis of changes in the representation of shRNAs will indicate critical Notch1 effectors. For in vitro validation of candidate genes, MM cell lines will be treated with target gene‐specific shRNAs or chemical inhibitors in combination with chemotherapeutic agents. Analyses of proliferation, cell cycle progression and apoptosis will reveal candidate genes, which contribute to growth and survival advantages. Most promising genes will be further tested in vivo using the MM mouse models BALB/c‐MOPC315.BM and VK∗MYC.Results:RNAseq analysis revealed 842 Notch1‐regulated genes. 40 downregulated and 28 upregulated genes were chosen as targets for the shRNA libraries. Specificity of the RNAseq approach was verified by quantitative PCR for known Notch1 target genes such as HEY1 and HES6. Interestingly, the group of Notch1‐upregulated genes further includes bone‐related genes supporting MM growth and the aggressive MM bone phenotype such as RUNX2, osteocalcin or cathepsin K (Trotter et. al., Blood, 2015).Expression of NIC and NΔE in engineered U266 cells was validated by flow cytometric and western blot analysis. Cell viability analysis by Annexin V/PI staining and CellTiter‐Glo® luminescent assay showed reduced drug sensitivity of Notch1‐expressing U266 cells compared to Notch1‐negative U266 cells, confirming the role of Notch1 as survival factor.Based on these results, the first functional screen was recently performed. Intermediate analysis of cell viability clearly showed a sensitizing effect of shRNAs, indicating promising screen hits. First outcomes of the screen will be presented at the meeting.Summary/Conclusion:ShRNA‐based functional HT screening is a potent tool for unveiling Notch1 downstream effectors as specific therapeutic targets in MM to improve treatment efficacy and reduce side effects.

Notch1 signaling in melanoma cells promoted tumor-induced immunosuppression via upregulation of TGF-\u03b21

BackgroundThe receptors of Notch family play an important role in controlling the development, differentiation, and function of multiple cell types. The aim of this study is to investigate the role of Notch1 signaling upon immune suppression induced by melanoma cells.MethodsMelanoma cell line B16 cells were transfected by lentivirus containing mouse Notch1 gene or Notch1 shRNA to generate B16 cell line that highly or lowly expressed Notch1. Notch1 in anti-tumor immune response was comprehensively appraised in murine B16 melanoma tumor model in immunocompetent and immunodeficient mice. The ratios of CD3+CD8+ cytotoxic T cells, CD49b+NK cells, CD4+CD25+FoxP3+ Tregs and Gr1+CD11b+ MDSCs in tumor-DLN or spleen were examined by flow cytometry. After the co-culture of B16 cells and CD8+ T cells, the effects of Notch1 on the proliferation and activation of T cells were assessed by CCK8 assay, CFSE dilution and Chromium-release test. The mRNA expression and supernatant secretion of immunosuppressive cytokines, TGF-β1, VEGF, IL-10 and IFN-γ were measured by RT-PCR and ELISA, respectively.ResultsDownregulation or overexpression of Notch1 in B16 melanoma cells inhibited or promoted tumor growth in immunocompetent mice, respectively. Notch1 expression in B16 melanoma cells inhibited the infiltration of CD8+ cytotoxic T lymphocytes and NK cells and reduced IFN-γ release in tumor tissue. It could also enhance B16 cell-mediated inhibition of T cell proliferation and activation, and upregulate PD-1 expression on CD4+ and CD8+ T cells. The percentage of CD4+CD25+FoxP3+ Tregs and Gr1+CD11b+MDSCs were significantly increased in tumor microenvironment, and all these were attributed to the upregulation of TGF-β1.ConclusionThese findings suggested that Notch1 signaling in B16 melanoma cells might inhibit antitumor immunity by upregulation of TGF-β1.

Risperidone ameliorates cognitive deficits, promotes hippocampal proliferation, and enhances Notch signaling in a murine model of schizophrenia

Antipsychotic agents have been reported to promote hippocampal neurogenesis and improve cognitive deficits; yet, the molecular mechanisms underlying these actions remain unclear. In the present study, we used a murine model of schizophrenia induced by 5-day intraperitoneal injection with the non-competitive N-methyl-d-aspartate receptor antagonist MK801 (0.3mg/kg/day) to assess cognitive behavioral deficits, changes in Notch signaling, and cellular proliferation in the hippocampus of adult male C57BL/6 mice after 2-week administration of risperidone (Rip, 0.2mg/kg/day) or vehicle. We then utilized in vivo stereotaxic injections of a lentivirus expressing a short hairpin RNA (shRNA) for Notch1 into the dentate gyrus to examine the role of Notch1 in the observed actions of Rip. We found that Rip ameliorated cognitive deficits and restored cell proliferation in MK801-treated mice in a manner associated with the up-regulation of Notch signaling molecules, including Notch1, Hes1, and Hes5. Moreover, these effects were abolished by pretreatment with Notch1 shRNA. Our results suggest that the ability of Rip to improve cognitive function in schizophrenia is mediated in part by Notch signaling.

MiR-34a inhibits pancreatic cancer progression through Snail1-mediated epithelial\u2013mesenchymal transition and the Notch signaling pathway

Epithelial–mesenchymal transition (EMT) and Notch signaling are important for the growth and invasion of pancreatic cancer, which is a leading cause of cancer-related deaths worldwide. miR-34a has been shown to play pivotal roles in the progression of several types of cancer. However, little is known about the regulatory mechanisms of miR-34a in pancreatic cancer processes. The aim of this study was to determine whether miR-34a has negative effects on pancreatic cancer and whether these effects are related to EMT and Notch signaling. In vitro, we demonstrated that miR-34a inhibited, while miR-34a inhibitors enhanced, migration and invasion of pancreatic cancer cell lines (PANC-1 and SW-1990).These effects were reversed by Snail1 overexpression or Snail1 shRNA. Furthermore, the anti-apoptotic effects of the miR-34a inhibitors in pancreatic cancer cells were abrogated by Notch1 shRNA. Luciferase reporter assays revealed that the Snail1 and Notch1 genes were direct targets of miR-34a. In vivo, we also demonstrated that miR-34a inhibited pancreatic cancer growth by decreasing Snail1 and Notch1 expression. Therefore, our results indicate that miR-34a inhibits pancreatic cancer progression by post-transcriptionally regulating Snail1 and Notch1 expression.

Downregulation of Notch1 induces apoptosis and inhibits cell proliferation and metastasis in laryngeal squamous cell carcinoma.

Notch signaling plays a key role in a wide variety of human tumors; it can be an oncogene or a tumor-suppressor gene depending on the tissue context. The functions of Notch1 in laryngeal squamous cell carcinoma (LSCC) are largely unknown. We investigated the role of Notch1 in LSCC cell growth, apoptosis and metastasis. We analyzed Notch1 expression in clinical LSCC samples using quantum dot immunohistochemistry (QD-IHC) and conventional IHC. Human laryngeal carcinoma HEp-2 cells were transfected with Notch1-specific short hairpin RNA (shRNA), and cell proliferation, apoptosis, and migration and invasion were evaluated using the cell counting assay, flow cytometry and wound healing and Transwell assays, respectively; western blotting was used to validate the expression of Notch1 target genes. Compared with normal tissues, Notch1 was upregulated in LSCC tissues; compared with LSCC tissues without metastasis, Notch1 upregulation was enhanced in LSCC tissues with metastasis (P<0.05). Transfection downregulated Notch1 mRNA and protein expression levels in the Notch1 shRNA group. There was a significantly greater decrease in cell proliferation in the Notch1 shRNA group than cell proliferation in the non-transfected (P<0.05) and negative shRNA groups (P<0.05). Furthermore, Notch1 knockdown induced apoptosis in the HEp-2 cells. Additionally, the number of migrated and invasive cells in the Notch1 shRNA group was decreased (P<0.05). Notch1 knockdown in the HEp-2 cells greatly inhibited phosphorylated extracellular signal-related kinase (p-ERK), phosphorylated protein kinase B (p-AKT), c-Myc, Bcl-2, p21, cyclin D1, cyclin-dependent kinase 4 (CDK4) and cyclin E expression levels and increased Bax expression. Altogether, our findings indicate that Notch1 expression is increased in human LSCC and correlates with tumorigenesis and metastasis, while in HEp-2 cells, Notch1 knockdown inhibited cell growth, induced apoptosis and inhibited migration and invasion by regulating Notch1 target genes, suggesting it may be a potential therapeutic target for treating LSCC.

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

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

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

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

Multifunctional core/shell nanoparticles cross-linked polyetherimide-folic acid as efficient Notch-1 siRNA carrier for targeted killing of breast cancer.

In gene therapy, how genetic therapeutics can be efficiently and safely delivered into target tissues/cells remains a major obstacle to overcome. To address this issue, nanoparticles consisting of non-covalently coupled polyethyleneimine (PEI) and folic acid (FA) to the magnetic and fluorescent core/shell of Fe3O4@SiO2(FITC) was tested for their ability to deliver Notch-1 shRNA. Our results showed that Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanoparticles are 64 nm in diameter with well dispersed and superparamagnetic. These nanoparticles with on significant cytotoxicity are capable of delivering Notch-1 shRNA into human breast cancer MDA-MB-231 cells with high efficiency while effectively protected shRNA from degradation by exogenous DNaseI and nucleases. Magnetic resonance (MR) imaging and fluorescence microscopy showed significant preferential uptake of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex by MDA-MB-231 cells. Transfected MDA-MB-231 cells exhibited significantly decreased expression of Notch-1, inhibited cell proliferation, and increased cell apoptosis, leading to the killing of MDA-MB-231 cells. In light of the magnetic targeting capabilities of Fe3O4@SiO2(FITC)/PEI-FA, our results show that by complexing with a second molecular targeting therapeutic, such as Notch-1 shRNA in this report, Fe3O4@SiO2(FITC)/PEI-FA can be exploited as a novel, non-viral, and concurrent targeting delivery system for targeted gene therapy as well as for MR imaging in cancer diagnosis.

Abstract 1952: Notch-1 regulated stemness and EMT in colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer death nationwide. In 2013, nearly 156,000 patients will be diagnosed with CRC. Surgical resection for early stage disease offers the best chance for cure; however, up to 30% of patients will relapse. Current models of CRC progression hold that tumor development is driven by a small subpopulation of cancer initiating (CIC) or cancer stem (CSC) cells that share many properties of stem cells. These cells have the capacity to drive tumor growth, are largely responsible for relapse and have been implicated in metastasis via epithelial to mesenchymal transition (EMT), similar to normal counterparts in organogenesis. Notch-1 is the major receptor for a highly conserved signaling pathway that is well-documented to control stem cell and lineage commitment in various organs, including the colon. The function of Notch signaling in colorectal cancer, however, is not well-defined. Based on preliminary data in which colon tumor samples from CRC patients expressed Notch-1 but surrounding benign tissue did not, we sought to test the hypothesis that Notch-1 signaling in colon cells promotes tumorigenesis by enhancing stem cell and EMT characteristics of tumor cells. The human colon tumor line HCT-116 was transduced with a retroviral construct that expresses constitutively active Notch-1 (ICN1). Hes-1 protein levels were increased in the activated Notch-1 transduced cells, indicating that the construct was functional in activating Notch-signaling. These HCT116/ICN1 cells exhibited higher plating efficiency than the parental cell line. The average colony size of the HCT116/ICN1 cells was about two-fold smaller than the parental cell line (p&amp;lt;0.001). In addition, cells with constitutively active Notch-1 (HCT116/ICN1) had a longer duplication time than parental HCT-116 cells (p&amp;lt;0.03). Consistent with longer duplication time, HCT116/ICN1 cells were two-fold slower in completing wound healing as compared with the parental HCT-116 cell line (p&amp;lt;0.001). Transwell migration assays revealed a two-fold increase in migration of HCT116/ICN1 cells as compared with the parental control (p&amp;lt;0.03). Western blot analysis demonstrated that the HCT116/ICN1 cells with activated Notch-1 expressed highly increased levels of the EMT associated proteins Smad3, CD44 and Slug. This was accompanied by a four-fold decreased expression of E-cadherin. Meanwhile, E-cadherin was not altered in HCT116 cells transduced with a Notch-1 shRNA lentiviral construct that inactivated Notch-1 signaling. Finally, levels of the numb protein, a Notch-1 antagonist, appeared to be significantly reduced (five-fold decrease) by constitutive Notch-1 signaling. Alternatively, inactivation of Notch-1 resulted in a four-fold increased expression of the numb protein. Collectively, our data point to the Notch-1 pathway as being a potential key regulator of EMT in colorectal tumors and as such, may be of therapeutic or prognostic utility. Citation Format: Alex Fender, Makenzie Nutter, Timothy Fitzgerald, Fred Bertrand, George Sigounas. Notch-1 regulated stemness and EMT in colorectal cancer. [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 1952. doi:10.1158/1538-7445.AM2014-1952

Notch1 is a potential therapeutic target for the treatment of human hepatitis B virus X protein-associated hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly lethal cancer with increasing worldwide incidence, and there are few therapeutics options available for patients with HCC. Thus, novel therapeutic targets for this disease are desperately needed. Chronic infection with hepatitis B virus (HBV) is the major risk factor for the development of HCC, while hepatitis B virus X protein (HBx) is essential for HBV-associated HCC. Based on our previous studies showing that HBx promoted hepatocarcinogenesis of the human non-tumor hepatic cell line L02 and activated Notch1 signaling, Notch1 short hairpin RNA (shRNA) was utilized to inhibit Notch1 mRNA in the present study. We observed that Notch1 shRNA inhibited cell proliferation together with decreased activity of the Notch1 pathway in vitro, and also markedly suppressed tumor formation of L02/HBx cells in a BALB/c nude mouse model in vivo. Furthermore, the blockade of Notch1 was capable of arresting the cell cycle in the G0/G1 phase through the downregulation of CyclinD1, CDK4, E2F1 and the upregulation of p21 and Rb, while all of these factors were involved in the CyclinD1/CDK4 pathway. Inhibition of Notch1 by shRNA markedly promoted the apoptosis of L02/HBx cells via the caspase-9-caspase-3 pathway. These data suggest that inhibition of Notch1 impairs the growth of human HBx-transformed L02 cells, and Notch1 may be a putative therapeutic target for human HBx-associated HCC.

Endothelial Differentiation of Adipose Tissue-Derived Mesenchymal Stromal Cells in Glioma Tumors: Implications for Cell-Based Therapy

Multipotent human adipose tissue mesenchymal stromal cells (hAMSCs) are promising therapy vehicles with tumor-homing capacity that can be easily modified to deliver cytotoxicity activating systems in the proximity of tumors. In a previous work, we observed that hAMSCs are very effective delivering cytotoxicity to glioma tumors. However, these results were difficult to reconcile with the relatively few hAMSCs surviving implantation. We use a bioluminescence imaging (BLI) platform to analyze the behavior of bioluminescent hAMSCs expressing HSV-tTK in a U87 glioma model and gain insight into the therapeutic mechanisms. Tumor-implanted hAMSCs express the endothelial marker PECAM1(CD31), integrate in tumor vessels and associate with CD133-expressing glioma stem cells (GSC). Inhibition of endothelial lineage differentiation in hAMSCs by Notch1 shRNA had no effect on their tumor homing and growth-promoting capacity but abolished the association of hAMSCs with tumor vessels and CD133+ tumor cells and significantly reduced their tumor-killing capacity. The current strategy allowed the study of tumor/stroma interactions, showed that tumor promotion and tumor-killing capacities of hAMSCs are based on different mechanisms. Our data strongly suggest that the therapeutic effectiveness of hAMSCs results from their association with special tumor vascular structures that also contain GSCs.

Role of Notch1 receptor on apoptosis of renal tubular epithelial cells transfected with HBx gene

To explore the expression of Notch1 receptor in renal tubular epithelial cells transfected with HBx gene and elucidate its role in cell apoptosis. The eukaryotic vector pcDNA3.1/myc-HBx containing HBx gene or vector pcDNA3.1/myc-Notch1 containing Notch1 gene was transiently transfected into HK-2 cells and shRNA technique employed for silencing Notch1. HK-2 cells were divided into 7 groups of normal culture, pcDNA3.1/myc, HBx, HBx + pcDNA3.1/myc, HBx + Notch1 gene, HBx + shRNA and HBx + Notch1 shRNA. Real-time polymerase chain reaction (PCR) and Western blotting were used to confirm the expression of HBx and detect the expression of Notch1 receptor. Methyl thiazolyl tetrazolium (MTT) assay was used to observe the proliferation rate of HK-2 cells and flow cytometry to detect the apoptosis of HK-2 cells. HBx and Notch1 receptor were successfully expressed in HK-2 cells post-transfection. The transfection efficiency of shRNA was 70%. The expression of Notch1 receptor in pcDNA3.1/myc-HBx group was higher than that in the normal culture and pcDNA3.1/myc groups. And the apoptotic ratio was also higher than that of normal culture and pcDNA3.1/myc group. The difference was significant (14.94% ± 0.32% vs 13.86% ± 0.18%, 13.67% ± 0.54%, both P < 0.05). The apoptotic ratio in the HBx + Notch1 gene group was lower than that in control group (10.10% ± 0.26% vs 14.79% ± 0.02%, P < 0.05). And the growth inhibition ratio of cells was also lower. But the apoptotic ratio and growth inhibition ratio were both higher in HBx + Notch1 shRNA group than those in HBx + shRNA group (both P < 0.05). HBx gene is successfully transfected into HK-2 cells. And its over-expression may induce apoptosis of HK-2 cells and growth inhibition by regulating Notch1 signal.

Notch1 signaling contributes to the oncogenic effect of HBx on human hepatic cells

Hepatocellular carcinoma (HCC) is a malignant tumor and hepatitis B virus X protein (HBx) plays a crucial role in its pathogenesis. The Notch1 signaling pathway is involved in various malignant tumors including liver cancers and down-regulation of Notch-1 may exert anti-tumor effects. Here, we demonstrate that inhibition of Notch1 by plasmid-based shRNA suppresses growth of human hepatic cells transfected with HBx through G0/G1 cell cycle arrest and apoptosis inhibition, possibly linked to the promoted expression of cyclin-dependent kinase inhibitor, P16, and decreased expression of apoptosis inhibitor, Bcl-2. The anti-proliferative and pro-apoptotic effects of Notch1 shRNA in HBx-transformed L02 cell may be partly mediated by down-regulation of nuclear factor-kappaB (NF-κB) binding activities, demonstrating possible cross-talk between Notch-1 and NF-κB signaling pathways. The oncogene HBx may therefore induce malignant transformation of human hepatic cells via Notch1 pathway, indicating that Notch1 plays a crucial role in HBx-related liver cancer and could be an effective therapeutic target for HCC.